Compositions of efavirenz

ABSTRACT

The present inventions relates to a solid composition and an aqueous dispersion comprising nanoparticles of the anti-retroviral drug efavirenz. The solid composition and aqueous dispersion additionally comprise a mixture of a hydrophilic polymer and a surfactant. The surfactant is selected from vitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammonium chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate, polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA), and a block copolymer of polyoxyethylene and polyoxypropylene, or a combination thereof. The hydrophilic polymer is suitably selected from polyvinyl alcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft copolymer, a block copolymer of polyoxyethylene and polyoxypropylene, polyethylene glycol, hydroxypropyl methyl cellulose (HPMC), and polyvinylpyrrolidone, or a combination thereof. The present invention also relates to processes for preparing both the solid composition and the aqueous dispersion, as well as to their use in therapy for the treatment and/or prevention of retroviral infections such as human immunodeficiency virus (HIV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry under 35 USC §371(b) of PCTInternational Application No. PCT/GB2012/052208, filed Sep. 7, 2012,which claims priority to United Kingdom Patent Application No.1115633.8, filed Sep. 9, 2011, the entire disclosures of all of whichare incorporated herein by reference.

INTRODUCTION

The present invention relates to compositions of the anti-HIV drugefavirenz that are suitable for pharmaceutical use. More specifically,the present invention relates to a solid composition of efavirenz and,in another aspect, to an aqueous dispersion of efavirenz. The presentinvention also relates to processes for preparing both the solidcomposition and the aqueous dispersion, as well as to their use intherapy for the treatment and/or prevention of retroviral infectionssuch as human immunodeficiency virus (HIV).

BACKGROUND OF THE INVENTION

Human Immunodeficiency Virus (HIV) is a major cause of morbidity andmortality in both the developed and the developing world. HIV is aretrovirus that causes acquired immunodeficiency syndrome (AIDS) inhumans, which in turn allows life-threatening infections and cancers tothrive as the immune system progressively fails.

HIV infection typically occurs through the transfer of bodily fluids,such as blood, semen, vaginal fluid, pre-ejaculate, or breast milk, fromone individual to another. HIV may be present within these bodily fluidsas either the free virus, or as a virus present within infected immunecells. HIV-1 tends to be the most virulent form of HIV, and istransmitted as a single-stranded enveloped RNA virus which, upon entryinto a target cell, is converted into double-stranded DNA by reversetranscription. This DNA may then become integrated into the host's DNAwhere it can reside in a latent from and avoid detection by the immunesystem. Alternatively, this DNA may be re-transcribed into RNA genomesand translated to form viral proteins that are released from cells asnew virus particles, which can then spread further.

A drug commonly used in the treatment of HIV, and particularly HIV-1, isefavirenz (EFV). Efavirenz is a non-nucleoside reverse transcriptaseinhibitor (NNRTI) that is widely used in highly active antiretroviraltherapy (HAART). The (−)-efavirenz enantiomer is sold commercially underthe trade names Sustiva™ or Stocrin™. The structure of (−)efavirenz isshown below.

Although efavirenz is effective in prolonging life expectancy in HIVsuffers, there are a number of drawbacks associated with the currentlyavailable formulations of efavirenz.

Efavirenz acts on an intracellular target, so the ability of efavirenzto penetrate into and accumulate within cells is a prerequisite foreffective treatment (Owen and Khoo, Journal of HIV Therapy, 2004, 9(4),53-57). One particular problem with the current formulations ofefavirenz is that the penetration of the drug into cells is variable andinadequate both within and between patients. As cellular penetration andaccumulation of the drug is necessary in order to effectively treat theHIV infected cells, there is a need for formulations of efavirenz thatexhibit good levels of cellular accumulation, particularly in immunecells (e.g. macrophages and CD4+ lymphocytes).

In addition, the distribution of efavirenz throughout the body is alsonot uniform with current efavirenz formulations, and certain targettissues and sanctuary sites, such as the brain and the testis, cansuffer from poor exposure to the drug. This can lead to sub-therapeuticlevels of the drug reaching certain tissues, with the consequentialeffect that HIV infected cells residing in these tissues may not beadequately treated. Furthermore, resistance to efavirenz is becoming anincreasing problem (Goshn et al., AIDS, 2009, 11, 165-173) and exposureto sub-therapeutic levels of efavirenz in these tissues increases therisk that efavirenz-resistant strains of HIV can arise and reseed theblood. Resistant strains of HIV are also transmittable meaning thatindividuals can be newly infected with resistant virus. There is,therefore, a need for formulations that provide an improved distributionof efavirenz throughout the body, and in particular to sanctuary sitesfor the virus.

A further problem with current efavirenz formulations is that it isnecessary to administer large doses of the drug each day (typically theadult dose is 600 mg per day), along with multiple other anti-HIV drugs.As a consequence, a patient will need to consume a large tablet orcapsule of efavirenz, or multiple smaller dosage tablets or capsules, inorder to obtain the required dosage. This can inevitably lead toproblems with patient compliance. Furthermore, efavirenz treatment isalso associated with a number of adverse side effects, which represent amajor problem for patients, especially over prolonged periods (Dahri andEnsom, Clin Pharmacokinet, 2007, 46(2), 109-132; Letendre et al,Conference Highlights—Neurologic Complications, 2010, 18(2), 45-55;Maggi et al, J. Antimicrob. Chemother., 2011, 66, 896-900). For thesereasons, there is a need for more effective formulations of efavirenz,which in turn may enable the required dosage of efavirenz to be lowered.Lower doses could have an effect on the number and/or size of thetablets/capsules that need to be consumed by the patient, as well asprevalence of the adverse side effects.

There is also a need for dosage forms that permit the dosage to beeasily varied on a patient-by-patient basis, depending on factors suchas the age (including paediatric dosing) and weight of the patient, aswell as the severity and stage of the infection.

It is therefore an object of the present invention to provide improvedformulations of efavirenz that address one or more of the drawbacksassociated with the current efavirenz formulations.

In particular, it is an object of the invention is to provide efavirenzformulations exhibiting good cell penetration and a more optimum andeffective distribution throughout the body.

Another object of the present invention is to provide efavirenzformulations with a high drug loading.

Another object of the present invention is to provide efavirenzformulations which permits lower overall dosage of efavirenz in HIVtreatments.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided asolid efavirenz composition, comprising nanoparticles of efavirenzdispersed within a mixture of at least one hydrophilic polymer and atleast one surfactant;

-   -   wherein the hydrophilic polymer is selected from polyvinyl        alcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft        copolymer, a block copolymer of polyoxyethylene and        polyoxypropylene, polyethylene glycol, hydroxypropyl methyl        cellulose (HPMC), and polyvinylpyrrolidone, or a combination        thereof; and    -   wherein the surfactant is selected from vitamin-E-polyethylene        glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene        sorbitan fatty acid ester, N-alkyldimethylbenzylammonium        chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,        polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA),        and a block copolymer of polyoxyethylene and polyoxypropylene,        or a combination thereof.

According to a second aspect of the present invention there is providedan aqueous dispersion, comprising a plurality of nanoparticles dispersedin an aqueous medium, the nanoparticles comprising a core of efavirenzand a coating of at least one hydrophilic polymer and at least onesurfactant;

-   -   wherein the hydrophilic polymer is selected from polyvinyl        alcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft        copolymer, a block copolymer of polyoxyethylene and        polyoxypropylene, polyethylene glycol, hydroxypropyl methyl        cellulose (HPMC), and polyvinylpyrrolidone, or a combination        thereof; and    -   wherein the surfactant is selected from vitamin-E-polyethylene        glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene        sorbitan fatty acid ester, N-alkyldimethylbenzylammonium        chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,        polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA),        and a block copolymer of polyoxyethylene and polyoxypropylene,        or a combination thereof.

Both the solid efavirenz composition and the aqueous dispersion of thepresent invention comprise the efavirenz drug substance in ananoparticulate form. The efavirenz nanoparticles of the presentinvention provide improved dosage forms of efavirenz, particularly withrespect to stability and efficacy. The solid efavirenz composition andthe aqueous dispersion of the present invention also provide good cellpenetration and accumulation, especially in immune cells. In certainembodiments, the level of cell penetration is significantly improvedrelative to conventional efavirenz formulations. The solid efavirenzcomposition and the aqueous dispersion of the present invention alsoprovide an improved distribution of drug throughout the body and providehigher drug levels in certain target tissues such as the testes andbrain. These advantages provide the opportunity for a more effectivetreatment of HIV and may also enable the required dosage of efavirenz tobe reduced.

The nanoparticles of efavirenz also possess low cytotoxicity andcompositions with high drug loadings can be prepared.

The provision of nanoparticles in a solid composition can also beadvantageous because it provides a more stable form of the drug that issuitable for long term storage. Furthermore, the solid composition canbe consumed as a solid dosage form when required in certain embodimentsof the invention, or, alternatively, they can be dispersed in a suitableaqueous diluent when required to form an aqueous dispersion of thenanoparticles for administration.

Furthermore, the solid compositions of the present invention allow forhigher drug loadings than known efavirenz formulations, which enablesexcipient dosages (e.g. surfactants) and the size of the dosage form tobe reduced.

Finally, the solid compositions of the present invention are ideallysuited to personalised medicine regimes, because the solid compositionsare substantially homogeneous, meaning that partial doses may beaccurately measured. Furthermore, the solid compositions of the presentinvention are readily dispersible within an aqueous medium to providestable aqueous dispersions. Such stable aqueous dispersions canthemselves be partitioned in a pre-determined manner to provide anaccurate liquid dose of efavirenz. Such methods of providingpersonalised doses are particularly applicable to paediatricadministration, since children require lower doses of efavirenz.Moreover, efavirenz doses can be accordingly adapted to suit a patient'sweight, age, and other circumstances (such as the stage or severity ofthe HIV infection).

According to a third aspect of the invention, there is provided anaqueous dispersion, obtainable by, obtained by, or directly obtained bydispersing the solid composition of the first aspect in an aqueousmedium.

According to a fourth aspect of the present invention there is providedprocesses for the preparation of a solid composition as defined herein.

According to a fifth aspect of the present invention, there is provideda solid composition obtainable by, obtained by, or directly obtained bythe process according to the fourth aspect.

According to a sixth aspect of the present invention, there is provideda pharmaceutical composition comprising a solid composition of the firstor fifth aspects of the invention, or an aqueous dispersion of thesecond or third aspects of the invention, and optionally a furtherpharmaceutically acceptable diluent, carrier, or excipient.

According to a seventh aspect of the present invention, there isprovided a solid composition or an aqueous dispersion as defined hereinfor use as a medicament.

According to a eighth aspect of the present invention, there is provideda solid composition or an aqueous dispersion as defined herein for usein the treatment and/or prevention of retroviral infections (e.g. HIV).

According to a ninth aspect of the present invention, there is provideda use of a solid composition or an aqueous dispersion as defined hereinin the manufacture of a medicament for use in the treatment and/orprevention of retroviral infections (e.g. HIV).

According to a tenth aspect of the present invention, there is provideda method of treating and/or preventing a retroviral infection (e.g.HIV), comprising administering a therapeutically effective amount of asolid composition, an aqueous dispersion, or a pharmaceuticalcomposition as defined herein to a patient suffering from or at risk ofsuffering from the retroviral infection.

According to an eleventh aspect of the present invention there isprovided a kit of parts comprising a solid composition as defined hereinor pharmaceutical composition comprising the solid composition asdefined herein, and a pharmaceutically acceptable diluent.

Features, including optional, suitable and preferred features of anyaspect of the invention are, unless otherwise stated, also features,including optional, suitable and preferred features of any other aspectof the invention.

BRIEF DESCRIPTION OF THE FIGURES

For a better understanding of the present invention, and to illustratehow embodiments of the same may be put into effect, reference is nowmade, by way of example, to the following figures in which:

FIG. 1 is a 3-D bar chart showing the results of a screen of sevenhydrophilic polymers with seven surfactants (see Example 5), with theclear “hits” (i.e. compositions satisfying the nanodispersion assessmentcriteria set out in Example 1) are shown as grey bars, near misses areshown as textured grey bars, more distant misses are shown astransparent bars, and non-hits are shown without bars.

FIG. 2 is a 3-D bar chart showing the results of a further screen of the6 clear “hits” identified from FIG. 1 but with different surfactantcontents (see Example 6). Again, the clear “hits” are shown as greybars, near misses are shown as textured grey bars, more distant missesare shown as transparent bars, and non-hits are shown without bars.

FIG. 3 shows (a) the z-average particle size (nm) for the original andrepeat samples of each of the 4 successful repeat samples; and (b) theaverage zeta potential (mV) for each of the 4 successful repeat samples(see Example 6).

FIG. 4 shows (a) the z-average particle size (nm) for low surfactant(5%) and high surfactant (10%) samples detailed in Table 6C; and (b) theaverage zeta potential (mV) for low surfactant (5%) and high surfactant(10%) samples detailed in Table 6C (see Example 6).

FIG. 5A shows time course measurements made upon nanodispersions of“cold” PVA/Vit-E samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/Vit-Esamples comprising both 5% (left hand side) and 10% (right hand size)surfactant content (see Example 7).

FIG. 5B shows time course measurements made upon nanodispersions of“cold” PVA/T-80 samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/T-80samples comprising both 5% (left hand side) and 10% (right hand size)surfactant content (see Example 7).

FIG. 5C shows time course measurements made upon nanodispersions of“cold” PVA/NDC samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/NDCsamples comprising both 5% (left hand side) and 10% (right hand size)surfactant content (see Example 7).

FIG. 6 is a graph showing how z-average particle size increases withincreased efavirenz loading (see Example 8).

FIG. 7 is a scatter chart showing data relating to the cytotoxicity ofthe various 10% drug loaded efavirenz formulations in terms of IC₅₀ (μM)across various cell lines. Data for aqueous efavirenz (i.e.non-nanodispersed) are given as dotted horizontal lines. Each circlerepresents a result for a particular nanodispersion (see Example 9).

FIG. 8 is a scatter chart showing activity of 10% loaded efavirenznanodispersions against recombinant HIV reverse transcriptase. Data foraqueous efavirenz are given as the dotted horizontal line. Again, eachcircle represents a result for a particular nanodispersion. Data aregiven as IC₅₀ in nM (see Example 10).

FIG. 9 is a scatter chart showing activity of 10% loaded efavirenznanodispersions against recombinant HIV reverse transcriptase. Data foraqueous efavirenz are given as the dotted horizontal line. Again, eachcircle represents a result for a particular nanodispersion. Data aregiven as IC₅₀ in nM (see Example 11).

FIG. 10 is a scatter chart showing cellular accumulation ratio(cell-associated drug divided by extracellular drug) of all 10% loadedefavirenz nanodispersions in the studied cell lines. Data for aqueousefavirenz are given as dotted horizontal lines. Again, each circlerepresents a result for a particular nanodispersion (see Example 12).

FIGS. 11A-E are bar charts showing cellular accumulation ratio(cell-associated drug divided by extracellular drug) of 70% loadedefavirenz nanodispersions in the studied cell lines, namely (A) HepG2,(B) Caco-2, (C) THP-1, (D) A-THP-1, (E) CEM, with respect to the sixnanodispersed efavirenz formulations (detailed in Table 8) and theparental aqueous drug (i.e. non-nanodisperse) sample (see Example 12).

FIG. 12 is a scatter chart showing apical (gut compartment) tobasolateral (blood compartment) transcellular permeability across Caco-2cell monolayers of 10% loaded efavirenz nanodispersions in the studiedcell lines. Data for aqueous efavirenz are given as dotted horizontallines. Again, each circle represents a result for a particularnanodispersion (see Example 13).

FIGS. 13A-F are a series of graphs showing transcellular permeability of70% efavirenz loaded nanodispersions across Caco-2 cell monolayersrelative to aqueous efavirenz. Data are given for apical to basolateraland for basolateral to apical passage of drug. Each panel (A-F)represent different 70% loaded nanodispersions of efavirenz (see Example13).

FIG. 14 shows pharmacokinetics of efavirenz and 70%-loaded efavirenznanodispersion in rats. A demonstrably higher plasma efavirenzconcentration was seen for the nanodispersion than for a conventionalformulation of efavirenz.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “nanoparticle” or “nanoparticulate” is used herein to mean aparticle having a size of less than or equal to 1 micron (μm).

The term “efavirenz” is used herein to refer to (−)-efavirenz, andincludes pharmaceutically acceptable salts and solvates thereof, as wellas any polymorphic or amorphous forms thereof.

It is to be appreciated that references to “preventing” or “prevention”relate to prophylactic treatment and includes preventing or delaying theappearance of clinical symptoms of the state, disorder or conditiondeveloping in a human that may be afflicted with or predisposed to thestate, disorder or condition but does not yet experience or displayclinical or subclinical symptoms of the state, disorder or condition.

It will be appreciated that references to “treatment” or “treating” of astate, disorder or condition includes: (1) inhibiting the state,disorder or condition, i.e., arresting, reducing or delaying thedevelopment of the disease or a relapse thereof (in case of maintenancetreatment) or at least one clinical or subclinical symptom thereof, or(2) relieving or attenuating the disease, i.e. causing regression of thestate, disorder or condition or at least one of its clinical orsubclinical symptoms.

A “therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the patientto be treated.

Solid Efavirenz Composition

The present invention provides a solid efavirenz composition, comprisingnanoparticles of efavirenz dispersed within a mixture of at least onehydrophilic polymer and at least one surfactant;

-   -   wherein the hydrophilic polymer is selected from polyvinyl        alcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft        copolymer, a block copolymer of polyoxyethylene and        polyoxypropylene, polyethylene glycol, hydroxypropyl methyl        cellulose (HPMC), and polyvinylpyrrolidone, or a combination        thereof; and    -   wherein the surfactant is selected from vitamin-E-polyethylene        glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene        sorbitan fatty acid ester, N-alkyldimethylbenzylammonium        chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,        polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA),        and a block copolymer of polyoxyethylene and polyoxypropylene,        or a combination thereof.

The nanoparticles of efavirenz drug substance are dispersed within asolid excipient mixture comprising the hydrophilic polymer and thesurfactant.

The solid composition of the present invention may be administered as itis to a patient, or further formulated to provide a pharmaceuticalcomposition in the form of, for example, a tablet, capsule, lozenge, ora dispersible powder or granule formulation.

The nanoparticles of efavirenz have an average particle size of lessthan or equal to 1 micron (μm). In a particular embodiment, thenanoparticles of efavirenz have an average particle size of between 100and 1000 nm. In another embodiment, the nanoparticles of efavirenz havean average particle size between 100 and 600 nm.

The particle size of the nanoparticles may be assessed by any suitabletechnique known in the art (e.g. laser diffraction, laser scattering,electron microscopy). In an embodiment of the invention, particle sizeis assessed by dispersing the solid composition in an aqueous medium anddetermining the particle size with a Zetasizer (Malvern InstrumentsLtd).

In an embodiment, the polydispersity of the nanoparticles of efavirenzis less than or equal to 0.8, suitably less than or equal to 0.6, andmost suitably less than or equal to 0.5. The polydispersity relates tothe size of the efavirenz nanoparticles and may be determined bysuitable techniques known in the art (e.g. laser diffraction, laserscattering, electron microscopy). In an embodiment of the presentinvention, the polydispersity of particle sizes of the nanoparticles ofefavirenz may be suitably assessed with a Malvern Zetasizer (MalvernInstruments Ltd).

In a particular embodiment, the average zeta potential of thenanoparticles of efavirenz when dispersed in an aqueous medium isbetween −100 and +100 mV. In another embodiment, the zeta potential ofthe nanoparticles of efavirenz is between −25 and +25 mV. In anotherembodiment, the zeta potential of the nanoparticles of efavirenz isbetween −20 and +20 mV. In general it has been found that zetapotentials of a relatively small magnitude (either positive or negative)allow the nanoparticles to better penetrate into and accumulate withincells. In accordance with the present invention, average zeta potentialscan be measured by techniques known in the art, such as using aZetasizer (Malvern Instruments Ltd).

The solid composition may comprise particles or granules of larger size,for example, 5 to 30 microns (μm) in size, but each particle or granulecontain a plurality of nanoparticles of efavirenz dispersed within amixture of the hydrophilic polymer and surfactant. Furthermore, theselarger particles or granules disperse when the solid composition ismixed with an aqueous medium to form discrete nanoparticles ofefavirenz.

In an embodiment, the solid composition comprises a single hydrophilicpolymer and a single surfactant selected from those listed herein. In analternative embodiment, the solid composition comprises two or morehydrophilic polymers and/or two or more surfactant selected from thoselisted herein may be present.

Hydrophilic Polymer

A wide range of hydrophilic polymers are suitable for use inpharmaceutical formulations. Examples of such polymers include:

(a) homo or co-polymers of monomers selected from: vinyl alcohol,acrylic acid, methacrylic acid, acrylamide, methacrylamide, acrylamideaminoalkylacrylates, aminoalkyl-methacrylates, hydroxyethylacrylate,methylpropane sulphonates, hydroxyethylmethylacrylate, vinylpyrrolidone, vinyl imidazole, vinyl amines, vinyl pyridine,ethyleneglycol, propylene glycol, ethylene oxides, propylene oxides,ethyleneimine, styrenesulphonates, ethyleneglycolacrylates andethyleneglycol methacrylate;(b) polyvinyl alcohol (PVA), a polyvinyl alcohol-polyethylene glycolgraft copolymer, a block copolymer of polyoxyethylene andpolyoxypropylene, polyethylene glycol, and polyvinylpyrrolidone, or acombination thereof(c) cellulose derivatives for example cellulose acetate,methylcellulose, methyl-ethylcellulose, hydroxy-ethylcellulose,hydroxy-ethylmethyl-cellulose, hydroxy-propylcellulose (HPC),hydroxy-propylmethylcellulose (HPMC), hydroxy-propylbutylcellulose,ethylhydroxy-ethylcellulose, carboxy-methylcellulose and its salts (egthe sodium salt—SCMC), or carboxy-methylhydroxyethylcellulose and itssalts (for example the sodium salt)(d) gums such as guar gum, alginate, locust bean gum and xanthan gum(e) polysaccharides such as dextran, xyloglucan and gelatin (orhydrolysed gelatin);(f) cyclodextrins such as beta-cyclodextrin;(g) mixtures thereof.

Copolymers may be statistical copolymers (also known as a randomcopolymer), a block copolymer, a graft copolymer or a hyperbranchedcopolymer. Additional co-monomers may also be present provided thattheir presence does not effect the water-solubility of the resultingpolymeric material.

Particular examples of homopolymers include poly-vinylalcohol (PVA),poly-acrylic acid, poly-methacrylic acid, poly-acrylamides (such aspoly-N-isopropylacrylamide), poly-methacrylamide; poly-acrylamines,poly-methyl-acrylamines, (such as polydimethylaminoethylmethacrylate andpoly-N-morpholinoethylmethacrylate), polyvinylpyrrolidone (PVP),poly-styrenesulphonate, polyvinylimidazole, polyvinylpyridine,poly-2-ethyl-oxazoline poly-ethyleneimine and ethoxylated derivativesthereof.

In the present invention, the hydrophilic polymer is selected from thosehydrophilic polymers that are capable of stabilising nanoparticles ofefavirenz in an aqueous dispersion together with a surfactant as definedherein, and which are also suitable for pharmaceutical use (e.g. theyare approved for use by the US Food and Drug Administration).

The hydrophilic polymer is therefore suitably selected from polyvinylalcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft copolymer,a block copolymer of polyoxyethylene and polyoxypropylene, polyethyleneglycol, hydroxypropyl methyl cellulose (HPMC), and polyvinylpyrrolidone,or a combination thereof.

It shall be appreciated that any molecular weight (Mw) or molecularnumber (Mn) values quoted herein span the range of Mw and Mn values thatmay be present in the polymer.

In a particular embodiment, the polyvinyl alcohol has an averagemolecular weight between 5000 and 200000 Da, suitably with a 75-90%hydrolysis level (i.e. % free hydroxyls). In a particular embodiment,the polyvinyl alcohol has a 75-90% hydrolysis level. In anotherembodiment, the polyvinyl alcohol has a 75-85% hydrolysis level. In aparticular embodiment, the polyvinyl alcohol has an average molecularweight between 9000 and 10000 Da, suitably with an 80% hydrolysis level.

In a particular embodiment, the polyvinyl alcohol-polyethylene glycolgraft copolymer has an average molecular weight between 30000 and 60000Da, suitably with a PVA/PEG ratio of between 90:10 and 25:75. In aparticular embodiment, the polyvinyl alcohol-polyethylene glycol graftcopolymer has an average molecular weight between 40000 and 50000 Da,suitably with a PVA/PEG ratio of between 85:15 and 25:75. Suitably thepolyvinyl alcohol-polyethylene glycol graft copolymer has a PVA/PEGratio of between 90:10 and 25:75, more suitably a PVA/PEG ratio ofbetween 85:15 and 25:75. In a particular embodiment, the polyvinylalcohol-polyethylene glycol graft copolymer is a Kollicoat® polymer(supplied by BASF). In a particular embodiment, the Kollicoat® isKollicoat® Protect.

The block copolymer of polyoxyethylene and polyoxypropylene is suitablyeither a diblock copolymer of polyoxyethylene and polyoxypropylene or atriblock copolymer thereof. In a particular embodiment, the blockcopolymer of polyoxyethylene and polyoxypropylene is a Poloxamer.

A “poloxamer” is a non-ionic triblock copolymer comprising a centralhydrophobic chain of polyoxypropylene, and hydrophilic chains ofpolyoxyethylene either side of this central hydrophobic chain. A“poloxamer” is typically named with the letter “P” followed by threenumerical digits (e.g. P407), where the first two digits multiplied by100 gives the approximate molecular mass of the polyoxypropylene chain,and the third digit multiplied by 10 provides the percentagepolyoxyethylene content of the poloxamer. For example, P407 is apoloxamer having a polyoxypropylene molecular mass of about 4,000 g/moland a polyoxyethylene content of about 70%. Poloxamers are also known asPluronics®, as well as by several other commercial names.

The Poloxamer is suitably a pharmaceutically acceptable Poloxomer. In aparticular embodiment, the poloxamer is Poloxamer P407 or PoloxamerP188.

In a particular embodiment, the polyethylene glycol (PEG) has an averagemolecular weight of 500 to 20000 Da. In a particular embodiment, thepolyethylene glycol is PEG 1K (i.e. with an average molecular weight ofabout 1000 Da).

In a particular embodiment, the HPMC has an average molecular weight of10000 to 400000 Da. In a particular embodiment, the HPMC has an averagemolecular weight of about 10000.

In a particular embodiment, the polyvinylpyrrolidone has an averagemolecular weight of 2000 to 1,000,000 Da. In a particular embodiment,the polyvinylpyrrolidone has an average molecular weight of 30000 to55000 Da. In a particular embodiment, the polyvinylpyrrolidone ispolyvinylpyrrolidone K30 (PVP K30).

In a particular embodiment, the hydrophilic polymer is selected fromPVA, a Kollicoat®, Poloxamer 407, PEG 1K, HPMC, PVP K30, and Poloxamer188, or a combination thereof.

In a particular embodiment, the hydrophilic polymer is selected from PVAor the polyvinyl alcohol-polyethylene glycol graft copolymer.

In a particular embodiment, the hydrophilic polymer is PVA.

Surfactant

Surfactants suitable for pharmaceutical use may be:

-   -   non-ionic (e.g. ethoxylated triglycerides; fatty alcohol        ethoxylates; alkylphenol ethoxylates; fatty acid ethoxylates;        fatty amide ethoxylates; fatty amine ethoxylates; sorbitan        alkanoates; ethylated sorbitan alkanoates; alkyl ethoxylates;        Pluronics™; alkyl polyglucosides; stearol ethoxylates; alkyl        polyglycosides; sucrose fatty acid esters, anionic, cationic,        amphoteric or zwitterionic);    -   anionic (e.g. alkylether sulfates; alkylether carboxylates;        alkylbenzene sulfonates; alkylether phosphates; dialkyl        sulfosuccinates; sarcosinates; alkyl sulfonates; soaps; alkyl        sulfates; alkyl carboxylates; alkyl phosphates; paraffin        sulfonates; secondary n-alkane sulfonates; alpha-olefin        sulfonates; isethionate sulfonates; alginates);    -   cationic (e.g. fatty amine salts; fatty diamine salts;        quaternary ammonium compounds; phosphonium surfactants;        sulfonium surfactants; sulfonxonium surfactants); or    -   zwitterionic (e.g. N-alkyl derivatives of amino acids (such as        glycine, betaine, aminopropionic acid); imidazoline surfactants;        amine oxides; amidobetaines).

Alkoxylated nonionic's (especially the PEG/PPG Pluronic™ materials),phenol-ethoxylates (especially TRITON™ materials), alkyl sulphonates(especially SDS), ester surfactants (preferably sorbitan esters of theSpan™ and Tween™ types) and cationics (especially cetyltrimethylammoniumbromide—CTAB) are particularly suitable as surfactants.

In the present invention, the surfactant is suitably selected from thosesurfactants that are capable of stabilising nanoparticles of efavirenztogether with a hydrophilic polymer as defined herein, and which arealso approved for pharmaceutical use (e.g. they are approved for use bythe US Food and Drug Administration).

The surfactant is therefore suitably selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), apolyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammoniumchloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA), and ablock copolymer of polyoxyethylene and polyoxypropylene, or acombination thereof.

It will be appreciated that the hydrophilic polymer and the surfactantmay both be either PVA or a block copolymer of polyoxyethylene andpolyoxypropylene. In other words, the PVA and block copolymer ofpolyoxyethylene and polyoxypropylene may function as both the surfactantand the hydrophilic polymer. The total amount of PVA or block copolymerof polyoxyethylene and polyoxypropylene that may be present in suchcircumstances is that defined hereinafter for the total of thesurfactant and hydrophilic polymer.

In a particular embodiment, the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), apolyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammoniumchloride, sodium deoxycholate, dioctyl sodium sulfosuccinate, andpolyethyleneglycol-12-hydroxystearate, or a combination thereof.

In a particular embodiment, the vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate) has a PEG moiety with an average molecular weightof 500 to 10000 Da. In a particular embodiment, thevitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate) has a PEGportion with an average molecular weight of about 1000 Da (which iscommercially available as Tocofersolan).

In a particular embodiment, the polyoxyethylene sorbitan fatty acidester is selected from Polysorbate 20 (commercially available as Tween®20) and Polysorbate 80 (commercially available as Tween® 80).

In a particular embodiment, the polyethyleneglycol-12-hydroxystearatehas a molecular weight of 300 to 3000 Da. In a particular embodiment,the polyethylenglycol-12-hydroxystearate has a molecular weight of 600to 700 Da (e.g. commercially available as Solutol® HS).

In a particular embodiment, the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate),Polysorbate 20, Polysorbate 80, N-alkyldimethylbenzylammonium chloride(e.g. commercially available as Hyamine®), sodium deoxycholate, dioctylsodium sulfosuccinate (e.g. AOT), andpolyethyleneglycol-12-hydroxystearate (e.g. Solutol® HS), or acombination thereof.

In an embodiment, the surfactant is selected from vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate), Polysorbate 20, Polysorbate 80,N-alkyldimethylbenzylammonium chloride, sodium deoxycholate, and dioctylsodium sulfosuccinate.

In a particular embodiment, the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate),Polysorbate 20, Polysorbate 80, N-alkyldimethylbenzylammonium chloride,and sodium deoxycholate.

In a particular embodiment, the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate),Polysorbate 80, N-alkyldimethylbenzylammonium chloride, and sodiumdeoxycholate.

In a particular embodiment, the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate),Polysorbate 80, and sodium deoxycholate.

In a particular embodiment, the surfactant is vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate).

Particular Combinations of Hydrophilic Polymer and Surfactant

PVA is a particularly suitable hydrophilic polymer where the surfactantis selected from vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate), Polysorbate 20, Polysorbate 80,N-alkyldimethylbenzylammonium chloride, sodium deoxycholate, and dioctylsodium sulfosuccinate.

PVA is a particularly suitable hydrophilic polymer where the surfactantis selected from vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate), Polysorbate 20, Polysorbate 80,N-alkyldimethylbenzylammonium chloride and sodium deoxycholate.

PVA is a particularly suitable hydrophilic polymer where the surfactantis selected from vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate), Polysorbate 80, N-alkyldimethylbenzylammoniumchloride and sodium deoxycholate.

PVA is a particularly suitable hydrophilic polymer where the surfactantis selected from vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate), Polysorbate 80, and sodium deoxycholate.

PVA is a particularly suitable hydrophilic polymer where the surfactantis vitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate).

The above PVA combinations are particularly advantageous where the PVAhas an average molecular weight between 9000 and 10000 Da, suitably witha 75-85% hydrolysis level.

The polyvinyl alcohol-polyethylene glycol graft copolymer (especiallyKollicoat®) is a particularly suitable hydrophilic polymer where thesurfactant is sodium deoxycholate.

Formulation of the Solid Composition

In a particular embodiment, the solid composition as defined hereincomprises 40 to 90 wt % of efavirenz. In another embodiment, the solidcomposition comprises 50 to 75 wt % of efavirenz. In another embodiment,the solid composition comprises 60 to 70 wt % of efavirenz.

The solid compositions of the present invention therefore permit highdrug loadings, which keeps the potentially toxic excipients (e.g.surfactants) to a minimum.

Suitably, the solid composition comprises 10 to 60 wt % of thehydrophilic polymer and surfactant combined, more suitably 20 to 60 wt%, even more suitably 25 to 50 wt %, most suitably 25 to 40 wt %. In aparticular embodiment, the solid composition comprises 25 to 35 wt % ofthe hydrophilic polymer and surfactant combined.

In a particular embodiment, the solid composition comprises 5 to 50 wt %of hydrophilic polymer. In another embodiment, the solid compositioncomprises 10 to 40 wt % of hydrophilic polymer. In another embodiment,the solid composition comprises 15 to 30 wt % of hydrophilic polymer. Ina particular embodiment, the solid composition comprises 15 to 25 wt %of hydrophilic polymer.

In a particular embodiment, the solid composition comprises 1 to 25 wt %of surfactant. In another embodiment, the solid composition comprises 2to 20 wt % of surfactant. In another embodiment, the solid compositioncomprises 3 to 10 wt % of surfactant.

Where either PVA or the block copolymer of polyoxyethylene andpolyoxypropylene serve both as the surfactant and the hydrophilicpolymer, the abovementioned wt % values for the hydrophilic polymer andsurfactant combined still apply. For example, where either PVA or theblock copolymer of polyoxyethylene and polyoxypropylene serve both asthe surfactant and the hydrophilic polymer, the solid compositionsuitably comprises 10 to 60 wt % of PVA or a block copolymer ofpolyoxyethylene and polyoxypropylene; more suitably 20 to 60 wt % of PVAor a block copolymer of polyoxyethylene and polyoxypropylene, even moresuitably 25 to 50 wt % of PVA or a block copolymer of polyoxyethyleneand polyoxypropylene, most suitably 25 to 40 wt % of PVA or a blockcopolymer of polyoxyethylene and polyoxypropylene. In a particularembodiment, the solid composition comprises 25 to 35 wt % of PVA or ablock copolymer of polyoxyethylene and polyoxypropylene.

In an embodiment, the solid composition comprises the hydrophilicpolymer and surfactant in a respective ratio of between 30:1 and 1:10.In a particular embodiment, the solid composition comprises thehydrophilic polymer and surfactant in a respective ratio of between 15:1and 1:2. In another embodiment, the solid composition comprises thehydrophilic polymer and surfactant in a respective ratio of between 10:1and 2:1. In a particular embodiment, the solid composition comprises thehydrophilic polymer and surfactant in a respective ratio of between 6:1and 3:1.

In a particular embodiment, the solid composition comprises:

-   -   40 to 80 wt % efavirenz;    -   10 to 40 wt % hydrophilic polymer; and    -   2 to 20 wt % surfactant.

In another embodiment, the solid composition comprises 11-29 wt % PVA asthe hydrophilic polymer, and 1-19 wt % vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate) as the surfactant.

In a particular embodiment, the solid composition comprises 15-25 wt %PVA as the hydrophilic polymer, and 5-15 wt % vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate) as the surfactant. In aparticular embodiment, the solid composition comprises 20-25 wt % PVA asthe hydrophilic polymer, and 5-10 wt % vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate) as the surfactant. In aparticularly preferred embodiment, the solid composition comprises 18-22wt % PVA as the hydrophilic polymer, and 8-12 wt %vitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate) as thesurfactant. Suitably, such compositions comprise 25 to 35 wt % of thehydrophilic polymer and surfactant combined, more suitably 28 to 32 wt%. In such embodiments, the solid composition suitably comprisesnanoparticles of efavirenz having an average particle size between 300and 400 nm, and an average zeta potential when dispersed in an aqueousmedium between −15 mV and −25 mV.

In a particular embodiment, the solid composition comprises 5-14 wt %PVA as the hydrophilic polymer, and 16-25 wt % Polysorbate 20 (e.g.Tween® 20) as the surfactant. Suitably such a composition comprises 25to 35 wt % of the hydrophilic polymer and surfactant combined.

In a particular embodiment, the solid composition comprises 16-29 wt %PVA as the hydrophilic polymer, and 1-14 wt % Polysorbate 80 (e.g.Tween® 80) as the surfactant. Suitably such a composition comprises 25to 35 wt % of the hydrophilic polymer and surfactant combined.

In a particular embodiment, the solid composition comprises 11-29 wt %PVA as the hydrophilic polymer, and 1-19 wt %N-alkyldimethylbenzylammonium chloride (e.g. Hyamine®) as thesurfactant. Suitably such a composition comprises 25 to 35 wt % of thehydrophilic polymer and surfactant combined.

In a particular embodiment, the solid composition comprises 21-29 wt %polyvinyl alcohol-polyethylene glycol graft copolymer (especiallyKollicoat®) as the hydrophilic polymer, and 1-9 wt % sodium deoxycholateas the surfactant. Suitably such a composition comprises 25 to 35 wt %of the hydrophilic polymer and surfactant combined.

In a particular embodiment, the solid composition comprises 11-29 wt %PVA as the hydrophilic polymer, and 1-19 wt % sodium deoxycholate as thesurfactant. Suitably such a composition comprises 25 to 35 wt % of thehydrophilic polymer and surfactant combined.

Unless otherwise stated, the above weight percentages relate to the % byweight of a particular constituent as a proportion of the total weightof the solid composition.

The solid composition may comprise one or more additional excipients,for instance, to further facilitate dispersion or stabilisation ofdispersions of the nanoparticles either in a pharmaceutically acceptablediluent or in vivo.

Processes for Preparing the Solid Composition

Solid compositions of the present invention may be prepared by a numberof methods well known in the art. Suitable techniques for forming suchcompositions are described in general terms in Horn and Reiger, Angew.Chem. Int. Ed., 2001, 40, 4330-4361.

For example, the solid composition may be prepared by milling a solidform of efavirenz. The milling may occur in the presence of thehydrophilic polymer and surfactant, or, alternatively, they may be mixedwith the milled drug after the milling step.

However, it is generally preferred that the solid efavirenz compositionsof the present invention are prepared by an oil in water emulsiontechnique whereby the efavirenz is dissolved in the oil phase and thehydrophilic polymer and surfactant are present in the water phase. Theoil and water solvents are then removed by freeze drying, spray dryingor spray granulation to provide a solid composition according to theinvention.

Thus, in accordance with the present invention, there is provided aprocess for preparing a solid composition as defined herein, the processcomprising:

-   -   (a) preparing an oil in water emulsion comprising:        -   an oil phase comprising efavirenz; and        -   an aqueous phase comprising a hydrophilic polymer and a            surfactant, each as defined herein; and    -   (b) removing the oil and water to form the solid composition.

An advantage of the process of the present invention is that theemulsions formed in step (a) are sufficiently homogenous and stable toallow for effective and uniform drying in step (b). Furthermore, thenanoparticles formed are substantially uniform in their physical form(size, shape etc.).

Step (a) may be performed by methods well-known in the art. Any suitablemethod for forming the oil in water emulsion defined in step (a) maytherefore be used. In particular, the mixing of the oil and water phasesto form the oil in water emulsion may be performed by methods well knownin the art. For example, the mixing may involve stirring, sonication,homogenisation, or a combination thereof. In a particular embodiment,the mixing is facilitated by sonication and/or homogenisation.

Step (a) may be performed, for example, by using the methods describedin WO 2004/011537 A1 (COOPER et al), which is hereby duly incorporatedby reference.

In a particular embodiment, step (a) comprises:

-   -   (i) providing an oil phase comprising efavirenz;    -   (ii) providing an aqueous phase comprising the hydrophilic        polymer and surfactant; and    -   (iii) mixing the oil phase and aqueous phase to produce the oil        in water emulsion.

Suitably, the oil phase is provided by dissolving efavirenz in asuitable organic solvent.

Suitably, the aqueous phase is provided by dissolving hydrophilicpolymer and surfactant in an aqueous medium, preferably in water.Alternatively the aqueous phase may be provided by mixing two separatelyprepared aqueous solutions of the surfactant and hydrophilic polymer.

In a particular embodiment, further aqueous medium (e.g. water) ororganic solvent is added before or during mixing step (iii).

The concentration of efavirenz in the oil in water emulsion is suitablyas concentrated as possible to facilitate effective scale-up of theprocess. For example, the concentration of efavirenz in the oil phase issuitably 20 mg/ml or higher, more suitably 40 mg/ml or higher, even moresuitably greater than 60 mg/ml or higher.

The concentration of the hydrophilic polymer in the aqueous/water phaseis suitably 0.5-50 mg/mL.

The concentration of the surfactant in the aqueous/water phase emulsionis suitably 0.5 to 50 mg/mL.

The organic solvent forming the oil phase is (substantially) immisciblewith water. Suitably the organic solvent is aprotic. Suitably theorganic solvent has a boiling point less than 120° C., suitably lessthan 100° C., suitably less than 90° C.

In a particular embodiment, the organic solvent is a selected from theClass 2 or 3 solvents listed in the International Conference onHarmonization (ICH) guidelines relating to residual solvents.

In a particular embodiment, the organic solvent is selected fromchloroform, dichloromethane, dichloroethane, tetrachloroethane,cyclohexane, hexane(s), isooctane, dodecane, decane, methylbutyl ketone(MBK), methylcyclohexane, tetrahydrofuran, toluene, xylene, butylacetate, mineral oil, tert-butylmethyl ether, heptanes(s), isobutylacetate, isopropyl acetate, methyl acetate, methylethyl ketone, ethylacetate, ethyl ether, pentane, and propyl acetate, or any suitablycombination thereof.

In a particular embodiment, the organic solvent is selected fromchloroform, dichloromethane, methylethylketone (MEK), methylbutylketone(MBK), and ethyl acetate.

The volume ratio of aqueous phase to oil phase in mixing step (iii) issuitably between 20:1 and 1:1, more suitably between 10:1 and 1:1, andmost suitably between 6:1 and 2:1.

Mixing step (iii) suitably produces a substantially uniform oil in wateremulsion. As previously indicated, mixing may be performed using methodswell known in the art. Suitably, mixing step (iii) involves stirring,sonication, homogenisation, or a combination thereof. In a particularembodiment, mixing step (iii) involves sonication and/or homogenisation.

Step (b) may be performed using methods well known in the art. Suitablystep (b) involves freeze drying, spray drying or spray granulation.

Step (b) may be performed using methods described in WO 2004/011537 A1(COOPER et al), the entire contents of which are hereby incorporated byreference.

In a particular embodiment, step (b) involves freeze drying the oil inwater emulsion. As such, step (b) may suitably comprise freezing the oilin water emulsion and then removing the solvents under vacuum.

Preferably, the freezing of the oil in water emulsion may be performedby externally cooling the oil in water emulsion. For example, a vesselcontaining the oil in water emulsion may be externally cooled, forexample, by submerging the vessel in a cooling medium, such as liquidnitrogen. Alternatively the vessel containing the oil in water emulsionmay be provided with an external “jacket” through which coolant iscirculated to freeze the oil in water emulsion. Alternatively the vesselmay comprise an internal element through which coolant is circulated inorder to freeze the oil in water emulsion.

In a further alternative, the oil in water emulsion is frozen by beingcontacted directly with a cooling medium at a temperature effective forfreezing the emulsion. In such cases, the cooling medium (e.g. liquidnitrogen) may be added to the oil in water emulsion, or the oil in wateremulsion may be added to the cooling medium. In a particular embodiment,the oil in water emulsion is added to the fluid medium (e.g. liquidnitrogen), suitably in a dropwise manner. This order of additionprovides higher purities of final product. As such, frozen droplets ofthe oil in water emulsion may suitably form. Such frozen droplets maysuitably be isolated (e.g. under vacuum to remove the fluidmedium/liquid nitrogen). The solvent is then suitably removed from thefrozen droplets under vacuum. The resulting solid composition is thenisolated.

In an alternative aspect, the present invention provides a process forpreparing a solid composition as defined herein, the process comprising:

-   -   preparing a single phase solution comprising efavirenz, a        hydrophilic polymer as defined herein, and a surfactant as        defined herein, in one or more solvents; and    -   spray-drying the mixture to remove the one or more solvents to        form the solid composition.

In this aspect of the invention, the single phase solution comprisingthe efavirenz, hydrophilic polymer, and surfactant are all dissolved inone solvent or two or more miscible solvents. Such processes are welldescribed in WO 2008/006712, the entire contents of which are dulyincorporated herein by reference. WO 2008/006712 also lists suitablesolvents and combinations thereof for forming the single phase solution.In an embodiment, the single phase solution comprises two or moresolvents (e.g. ethanol and water) which together solubilise efavirenz,hydrophilic polymer, and the surfactant. In another embodiment, thesingle phase comprises a single solvent, for example ethanol or water.Removing of the one or more solvents from the single phase fluid mixtureinvolves spray drying—again WO 2008/006712 details suitable spray-dryingconditions.

The present invention also provides a solid composition obtainable by,obtained by, or directly obtained by any of the processes describedherein.

Aqueous Dispersion

The present invention provides an aqueous dispersion, comprising aplurality of nanoparticles dispersed in an aqueous medium, thenanoparticles comprising a core of efavirenz and a coating of at leastone hydrophilic polymer and at least one surfactant;

-   -   wherein the surfactant is selected from vitamin-E-polyethylene        glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene        sorbitan fatty acid ester, N-alkyldimethylbenzylammonium        chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,        polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA),        and a block copolymer of polyoxyethylene and polyoxypropylene,        or a combination thereof.

The present invention further provides an aqueous dispersion, comprisinga plurality of nanoparticles dispersed in an aqueous medium, thenanoparticles comprising a core of efavirenz and a coating of at leastone hydrophilic polymer and at least one surfactant;

-   -   wherein the hydrophilic polymer is selected from polyvinyl        alcohol (PVA), a polyvinyl alcohol-polyethylene glycol graft        copolymer, a block copolymer of polyoxyethylene and        polyoxypropylene, polyethylene glycol, hydroxypropyl methyl        cellulose (HPMC), and polyvinylpyrrolidone, or a combination        thereof; and    -   wherein the surfactant is selected from vitamin-E-polyethylene        glycol-succinate (Vit-E-PEG-succinate), a polyoxyethylene        sorbitan fatty acid ester, N-alkyldimethylbenzylammonium        chloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,        polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA),        and a block copolymer of polyoxyethylene and polyoxypropylene,        or a combination thereof.

The present invention also provides an aqueous dispersion, obtainableby, obtained by, or directly obtained by dispersing the solidcomposition as defined herein in an aqueous medium. Suitably, an aqueousdispersion is prepared immediately prior to use.

When the solid composition is dispersed in the aqueous medium, thehydrophilic polymer and/or surfactant is dissolved within the aqueousmedium to release the nanoparticles of efavirenz in a dispersed form.The nanoparticles of efavirenz, which were formerly dispersed within asolid mixture of the hydrophilic polymer and surfactant, then becomedispersed within the aqueous medium in a coated form, whereby the coreof efavirenz is coated with the hydrophilic polymer and surfactant. Sucha coating is thought to impart stability to the nanoparticles, therebypreventing premature coagulation and aggregation.

Suitably the relative amounts (including ratios) of efavirenz,hydrophilic polymer, and surfactant are the same as defined in relationto the solid composition. However, their respective wt % values in theaqueous dispersion as a whole must be adjusted to take account of theaqueous medium. In a particular embodiment, the aqueous medium comprises20 to 99.5 wt % of the total aqueous dispersion. In a particularembodiment, the aqueous medium comprises 50 to 98 wt % of the totalaqueous dispersion. In a particular embodiment, the aqueous mediumcomprises 70 to 95 wt % of the total aqueous dispersion. Suitably, theremaining proportion of the aqueous dispersion essentially consists ofefavirenz, hydrophilic polymer, and surfactant, whose proportions withinthe aqueous dispersion as a whole are accordingly calculated (andscaled) by reference to the proportions recited in relation to the solidcomposition.

In a particular embodiment, the aqueous medium is water. In analternative embodiment, the aqueous medium comprises water and one ormore additional pharmaceutically acceptable diluents or excipients.

Aqueous dispersions of the present invention are advantageously stablefor prolonged periods, both in terms of chemical stability and thestability of the particles themselves (i.e. with respect to aggregation,coagulation, etc.).

Aqueous dispersions of the present invention may be considered aspharmaceutical compositions of the present invention.

Aqueous dispersions of the present invention allow a measured aliquot tobe taken therefrom for accurate dosing in a personalised medicineregime.

The particle size, polydispersity and zeta potential of thenanoparticles of efavirenz in the aqueous dispersion is as definedhereinbefore in relation to the solid composition. It will of course beappreciated that the particle size, polydispersity and zeta potentialnanoparticles of efavirenz present in the solid composition are measuredby dispersing the solid composition in an aqueous medium to thereby forman aqueous dispersion of the present invention.

In an embodiment, the aqueous dispersion comprises a single hydrophilicpolymer and a single surfactant selected from those listed herein. In analternative embodiment, the aqueous dispersion comprises two or morehydrophilic polymers and/or two or more surfactants selected from thoselisted herein.

Process for Preparing an Aqueous Dispersion

The aqueous dispersion may be formed by methods well known in the art.For example, efavirenz may be milled in the presence of an aqueousmixture of the hydrophilic polymer and surfactant.

In a particular aspect of the invention, however, there is provided aprocess for preparing an aqueous dispersion, comprising dispersing asolid efavirenz composition as defined herein in an aqueous medium.

In a particular embodiment, the aqueous medium is water. In analternative embodiment, the aqueous medium comprises water and one ormore additional excipients.

Dispersing the solid composition in the aqueous medium may compriseadding the solid composition to an aqueous medium and suitably agitatingthe resulting mixture (e.g. by shaking, homogenisation, sonication,stirring, etc.).

Pharmaceutical Compositions

The present invention provides a pharmaceutical composition comprising asolid composition or an aqueous dispersion as defined herein. Thepharmaceutical compositions of the present invention may furthercomprise one or more additional pharmaceutically acceptable excipients.

The solid efavirenz compositions of the invention may be formulated intoa form suitable for oral use (for example as tablets, lozenges, hard orsoft capsules, or dispersible powders or granules) by techniques knownin the art. As such, the solid compositions of the invention may bemixed with one or more additional pharmaceutical excipients during thisprocess, such as antiadherants, binders, coatings, enterics,disintegrants, fillers, diluents, flavours, colours, lubricants,glidants, preservatives, sorbents, and sweeteners.

In a particular embodiment, the pharmaceutical composition is a tabletor capsule comprising the solid efavirenz composition.

The aqueous dispersion of the present invention may be administered asit is or further formulated with one or more additional excipients toprovide a dispersion, elixir or syrup that is suitable for a oral use,or a dispersion that is suitable for parenteral administration (forexample, a sterile aqueous dispersion for intravenous, subcutaneous,intramuscular, intraperitoneal or intramuscular dosing).

In a particular embodiment, the pharmaceutical composition is an aqueousdispersion as described herein. Such dispersed formulations can be usedto accurately measure smaller dosages, such as those suitable foradministration to children.

In a particular embodiment, the pharmaceutical composition is in a formsuitable for parenteral delivery, whether via intravenous orintramuscular delivery.

It will be appreciated that different pharmaceutical compositions of theinvention may be obtained by conventional procedures, using conventionalpharmaceutical excipients, well known in the art.

The pharmaceutical compositions of the invention contain atherapeutically effective amount of efavirenz. A person skilled in theart will know how to determine and select an appropriate therapeuticallyeffective amount of efavirenz to include in the pharmaceuticalcompositions of the invention.

Uses of the Nanoparticles Formulation and Pharmaceutical Composition

The present invention provides a solid composition or an aqueousdispersion as defined herein for use as a medicament.

The present invention further provides a solid composition or an aqueousdispersion as defined herein for use in the treatment and/or preventionof retroviral infections (e.g. HIV).

The present invention further provides a use of a solid composition oran aqueous dispersion as defined herein in the manufacture of amedicament for use in the treatment and/or prevention of retroviralinfections (e.g. HIV).

The present invention further provides a method of treating and/orpreventing a retroviral infection (e.g. HIV), the method comprisingadministering a therapeutically effective amount of a solid composition,an aqueous dispersion, or a pharmaceutical composition as definedherein, to a patient suffering from or at risk of suffering from aretroviral infection.

The term “retrovirus” generally refers to an RNA virus capable ofself-duplication in a host cell using the reverse transcriptase enzymeto transcribe its RNA genome into DNA. The DNA is then potentiallyincorporated into the host's genome so that the virus can then replicatethereafter as part of the host's DNA.

The retroviral infection to be treated or prevented is suitably selectedfrom human immunodeficiency virus (HIV), Alpharetrovirus,Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus,Lentivirus, Spumavirus, Metavirus, Errantvirus, Pseudovirus,Hepadnavirus, and Caulimovirus.

In a particular embodiment of the present invention, the retroviralinfection to be treated or prevented is the human immunodeficiency virus(HIV), most suitably the human immunodeficiency virus (HIV) type 1.

The solid compositions, aqueous dispersions, and pharmaceuticalcompositions of the present invention are suitably used as part ofhighly antiretroviral therapy (HAART) in the treatment of humanimmunodeficiency virus (HIV) type 1.

The solid compositions, aqueous dispersions, and pharmaceuticalcompositions of the present invention are also suitably used to reducethe risk of or prevent HIV infection developing in subjects exposed to arisk of developing HIV infection.

Efavirenz, the active agent in the solid compositions, aqueousdispersion, and pharmaceutical compositions of the present invention, isan antiretroviral drug which acts allosterically by binding the reversetranscriptase enzyme at a site distinct and distal to the active site,known as the non-nucleoside reverse transcriptase inhibitor (NNRTI)pocket. As such, the solid composition, aqueous dispersion, andpharmaceutical compositions of the present invention are capable ofinhibiting non-nucleoside reverse transcriptase activity. Moreover, thenanoparticles and pharmaceutical compositions of the present inventionare suitable for use in antiretroviral therapies and prophylactictreatments.

Thus in another aspect of the invention there is provided a method ofinhibiting non-nucleoside reverse transcriptase activity in a cell (invivo or in vitro), the method comprising administering to said cell asolid composition, aqueous dispersion, or pharmaceutical composition asdescribed herein.

In another aspect, the present invention provides a method of inhibitingnon-nucleoside reverse transcriptase activity in a human or animalsubject in need of such inhibition, the method comprising administeringto said subject an effective amount of a solid composition, aqueousdispersion, or pharmaceutical composition as defined herein.

In another aspect, the present invention provides a solid composition,aqueous dispersion, or pharmaceutical composition as defined herein foruse in the treatment of a disease or condition associated withnon-nucleoside reverse transcriptase activity.

In another aspect, the present invention provides the use of a solidcomposition, aqueous dispersion, or pharmaceutical composition asdefined herein in the manufacture of a medicament for use in thetreatment of a disease or condition associated with non-nucleosidereverse transcriptase activity.

Routes of Administration

The solid compositions, aqueous dispersions, and pharmaceuticalcompositions of the invention may be administered to a subject by anyconvenient route of administration.

Routes of administration include, but are not limited to, oral (e.g. byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, infraarterlal, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; or by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

In a particular embodiment (e.g. in HIV treatments), the route ofadministration is either oral or by implant of a depot or reservoirformulation.

Combination Therapy

Although it is possible that the solid compositions, aqueousdispersions, and pharmaceutical compositions of the invention may beused as a sole medicament in the treatment and/or prevention of aretrovirus infection such as HIV, it is more typical that this agentwill be used in combination with one or more additional anti-retroviraland/or anti-microbial agents.

Other antiretroviral agents suitable in combination treatments with theformulations and compositions of the present invention includeZidovudine, Zalcitabine, Didanosine, Stavudine, Lamivudine, Abacavir,Combivir (zidovudine+lamivudine), Trizivir(zidovudine+lamivudine+abacavir), Tenofovir, Emtricitabine, Truvada(Tenofovir+Emtricitabine), Epzicom/Kivexa (abacavir+lamivudine),Hydroxyurea, Nevirapine, Delavirdine, Etravirine, Rilpivirine, Atripla(efavirenz+emtricitabine+tenofovir), Indinavir, Ritonavir, Saquinavir,Nelfinavir, Amprenavir, Kaletra (lopinavir+ritonavir), Atazanavir,Fosamprenavir, Tipranavir, Darunavir, Enfuvirtide, Maraviroc,Raltegravir, or combinations thereof.

In a particular embodiment, the other suitable antiretroviral agentssuitable for use in combination treatments with the formulations andcompositions of the present invention include Tenofovir, Lamivudine,Abacavir, Emtricitabine, Zidovudine, Combivir (zidovudine+lamivudine),Truvada (Tenofovir+Emtricitabine), Epzicom/Kivexa (abacavir+lamivudine),Lopinavir, Ritonavir, and Kaletra (lopinavir+ritonavir), or combinationsthereof.

In particular embodiments, the other antiretroviral agents suitable incombination with formulations and compositions of the present inventionare themselves provided as combinations such as:

-   -   Emtricitabine+Tenofovir disoproxil fumarate    -   Lamivudine+Stavudine    -   Lamivudine+Tenofovir disoproxil fumarate    -   Lamivudine+Zidovudine    -   Lamivudine+Didanosine.

Accordingly, an aspect of the invention provides a combination suitablefor use in the treatment or prevention of a retrovirus infection, suchas HIV, comprising a solid composition, an aqueous dispersion, or apharmaceutical composition as defined hereinbefore, and one or moreother antiretroviral agents.

The present invention also provides a solid composition, an aqueousdispersion, or a pharmaceutical composition as defined hereinbefore foruse in the treatment or prevention of a retrovirus infection, such asHIV, wherein the solid composition, aqueous dispersion, orpharmaceutical composition is administered in combination with one ormore other antiretroviral agents.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a solid efavirenz composition as defined hereinand one or more additional antiretroviral agents.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising an aqueous dispersion as defined herein whichfurther comprises one or more additional antiretroviral agents.

Herein, where the term “combination” is used it is to be understood thatthis refers to simultaneous, separate or sequential administration. Inone aspect of the invention “combination” refers to simultaneousadministration. In another aspect of the invention “combination” refersto separate administration. In a further aspect of the invention“combination” refers to sequential administration. Where theadministration is sequential or separate, the delay in administering thesecond component should not be such as to lose the beneficial effect ofthe combination.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the formulations orcompositions of this invention within the dosage range describedhereinbefore and the other pharmaceutically-active agent within itsapproved dosage range.

In a further aspect of the invention, there is provided a pharmaceuticalcomposition comprising a solid composition or an aqueous dispersion asdefined herein; and one or more other antiretroviral agents. In aparticular embodiment, the pharmaceutical composition is a single dosageform.

Kit of Parts

The present invention provides a kit of parts comprising a solidcomposition as defined herein or pharmaceutical composition comprisingthe solid composition as defined herein, and a pharmaceuticallyacceptable aqueous diluent.

The solid composition or pharmaceutical composition comprising the solidcomposition as defined herein can be dispersed into the diluent toprovide an aqueous dispersion as defined herein. Either the entiredispersion can then be administered, or a proportion of it can bemeasured and then administered (thereby providing a means ofadministering different dosages to individual patients).

EXAMPLES Materials

All materials were purchased and used without further purification fromSigma-Aldrich unless specified otherwise.

Example 1 Screening for Nanoformulations—10 Hydrophilic Polymers, 16Surfactants

Samples are prepared using a 10 mgml⁻¹ stock solution of efavirenz (A)in chloroform, a 22.5 mgml⁻¹ of polymer (P) and a 22.5 mgml⁻¹ stocksolution of surfactant (S). Stock solutions are added in the followingproportion; 100 μl (A); 267 μl (P) and 133 μl (S), therefore solid massratio is; 10% (A), 60% (P) and 30% (S) in an 1:4 oil to water (0/W) mix.The mixtures are then emulsified using a probe sonicator (UP400Smanufactured by Hielscher (Germany)), fitted with an H3 titanium probe)operated at 20% amplitude for 7 seconds followed by immediate cryogenicfreezing.

A matrix of 160 samples (comprised of 10 different polymers and 16surfactants) was prepared. Once all 160 samples had been prepared, theywere lyophilised (Virtis benchtop K) for 42 hours to leave a dry porousproduct, the samples were then sealed in individual vials untilanalysis.

The polymers and surfactants employed in this screen are detailed inTable 1A and Table 1B below:

TABLE 1A List of hydrophilic polymers initially screened m/dm{circumflexover ( )}3 Polymer MW (22.5 mg/ml) PEG 1k 1000 0.00225 F68 84000.000267857 F127 12600 0.000178571 Kollicoat 45000 0.00005 PVA 95000.000236842 PVP k30 30000 0.000075 HPC 80000 0.000028125 HPMC 100000.000225 Hydrolysed 1982 0.001135217 gelatin NaCMC 90000 0.000025

TABLE 1B List of 16 Surfactants initially screened m/dm{circumflex over( )}3 Surfactant MW (22.5 mg/ml) Na alginate 155000 1.45161E-05 NaMyristate 250.35 0.008987418 Na Deoxycholate 414.55 0.005427572 NaCaprylate 166.19 0.013538721 Vit E-peg- 1000 0.00225 succinate Sisterna11 650 0.003461538 Sisterna 16 650 0.003461538 SDS 288.38 0.007802205AOT 444.56 0.005061184 Chremophor EL 2500 0.0009 Solutol HS 344.530.006530636 Tween 20 1227 0.001833741 Tween 80 1300 0.001730769 Brij 581123.52 0.002002635 Hyamine 448.08 0.005021425 CTAB 364.46 0.006173517Screen Analysis

Immediately prior to analysis, samples were dispersed by addition of 1ml of water. The particle size of the active, organic nanoparticulatedispersion is then measured by dynamic light scattering (DLS) using aMalvern Zetasizer Nano ZS. 3 measurements using automatic measurementoptimisation, Malvern Zetasizer software version 6.20 was used for dataanalysis. The particles were considered hits if the below criteria weremet.

Nanodispersion Quality Assessment Criteria

A particle is determined a hit if it complies with the followingcriteria: Complete dispersion of the sample with no large particlesvisible, a particle Z-average <1000 nm, a polydispersity index (PDI)<0.5, a standard deviation between three scans <10% from averageZ-average and two of the three DLS scans pass the size quality report.The size quality report incorporates 12 tests on the reliability of thedata recorded and is automatically applied to each measurement by theMalvern Zetasizer software. These tests ensure that the sample is withina size range appropriate for DLS, has a PDI below 1, is within thecorrect concentration range and that the cumulant and distribution fitare good (i.e. the errors on the data are less than 0.005).

Table 1C below lists the hits in terms of suitable hydrophilic polymersand surfactants.

TABLE 1C hits of suitable hydrophilic polymers and surfactants (66 hitsin all) Hydrophilic Polymer Surfactant PEG 1K Na Deoxycholate. PEG 1K Nacaprylate. PEG 1K Sisterna 16 PEG 1K SDS PEG 1K AOT PEG 1K Tween 20 PEG1K Tween 80 PEG 1K Brij 58 PEG 1K Hyamine Pluronic F68 Na alginate.Pluronic F68 Na caprylate Pluronic F68 Vit E-peg-succinate Pluronic F68Chremophor EL Pluronic F68 Tween 20 Pluronic F68 Tween 80 Pluronic F68Hyamine Pluronic F68 CTAB Pluronic F127 Na Deoxycholate Pluronic F127 Nacaprylate. Pluronic F127 Vit E-peg-succinate Pluronic F127 SDS PluronicF127 Tween 80 Pluronic F127 CTAB Kollicoat Na alginate. Kollicoat NaDeoxycholate. Kollicoat Na caprylate. Kollicoat Vit E-peg-succinateKollicoat Sisterna 11 Kollicoat Sisterna 16 Kollicoat AOT KollicoatTween 20 Kollicoat Tween 80 Kollicoat Brij 58 Kollicoat HyamineKollicoat CTAB PVA Na Deoxycholate. PVA Sisterna 11 PVA Tween 20 PVATween 80 PVA Hyamine PVA CTAB PVP 30K Vit E-peg-succinate PVP 30KChremophor EL PVP 30K Solutol HS PVP 30K Tween 20 PVP 30K Tween 80 PVP30K Brij 58 PVP 30K Hyamine PVP 30K CTAB HPMC Vit E-peg-succinate HPMCSolutol HS HPMC Tween 20 HPMC Tween 80 HPMC Brij 58 HPMC Hyamine HPMCCTAB Hydrolysed gelatine Na Deoxycholate Hydrolysed gelatine Nacaprylate. Hydrolysed gelatine Chremophor EL Hydrolysed gelatine SolutolHS Hydrolysed gelatine Tween 20 Hydrolysed gelatine Tween 80 Hydrolysedgelatine Brij 58 Hydrolysed gelatine Hyamine NaCMC Tween 20 NaCMC Brij58

Example 2 Efavirenz Low Surfactant Screen

A further 160 screen was prepared for EFV using a lower quantity ofsurfactant and a higher proportion of polymer. The samples were preparedby the identical method mentioned in Example 1, however ratios employedwere 100 μl (A), 333 μl (P) and 67 μl (S), therefore solid mass ratiois; 10% (A), 75% (P) and 15% (S) in an 1:4 oil to water (OW) mix. Allother processing, including screening, were the same as described inExample 1. The hits, which were determined in accordance with thenanodispersion quality assessment criteria described in Example 1, arelisted in Table 2 below.

TABLE 2 hits of suitable hydrophilic polymers and surfactants (77 hitsin all) Hydrophilic Polymer Surfactant PEG 1K Na alginate PEG 1K NaDeoxycholate. PEG 1K Na caprylate. PEG 1K Sisterna 16 PEG 1K SDS PEG 1KAOT PEG 1K Chremophor EL PEG 1K Solutol HS PEG 1K Tween 20 PEG 1K Tween80 PEG 1K Brij 58 PEG 1K Hyamine Pluronic F68 Na alginate. Pluronic F68Na caprylate Pluronic F68 AOT Pluronic F68 Chremophor EL Pluronic F68Solutol HS Pluronic F68 Hyamine Pluronic F68 CTAB Pluronic F127 Nacaprylate. Pluronic F127 Vit E-peg- succinate Pluronic F127 Tween 20Pluronic F127 Tween 80 Pluronic F127 CTAB Kollicoat Na alginate.Kollicoat Na caprylate. Kollicoat Vit E-peg- succinate KollicoatSisterna 11 Kollicoat Sisterna 16 Kollicoat SDS Kollicoat AOT KollicoatChremophor EL Kollicoat Solutol HS Kollicoat Tween 20 Kollicoat Tween 80Kollicoat Brij 58 Kollicoat Hyamine Kollicoat CTAB PVA Na alginate PVANa caprylate PVA Sisterna 11 PVA Chremophor EL PVA Solutol HS PVA Tween20 PVA Tween 80 PVA hyamine PVP 30K Na caprylate PVP 30K Vit E-peg-succinate PVP 30K AOT PVP 30K Chremophor EL PVP 30K Solutol HS PVP 30KTween 20 PVP 30K Tween 80 PVP 30K Brij 58 PVP 30K hyamine PVP 30K CTABHPMC Vit E-peg- succinate HPMC AOT HPMC Chremophor EL HPMC Solutol HSHPMC Tween 20 HPMC Tween 80 HPMC Brij 58 HPMC Hyamine HPMC CTABHydrolysed gelatine Na Deoxycholate Hydrolysed gelatine Na caprylate.Hydrolysed gelatine Vit E-peg- succinate Hydrolysed gelatine SDSHydrolysed gelatine Chremophor EL Hydrolysed gelatine Solutol HSHydrolysed gelatine Tween 20 Hydrolysed gelatine Brij 58 Hydrolysedgelatine Hyamine NaCMC Vit E-peg- succinate NaCMC Chremophor EL NaCMCBrij 58

Example 3 Nanoformulations Assessed in Biological Assays

‘Hits’ (i.e. those listed under Examples 1 and 2 in Tables 10 and 2)from the previous screen were remade in accordance with the proceduresdescribed in Example 1 in replicate (one sample to be characterised byDLS and zeta potential analysis, with an additional sample for eachbiological assay to be carried out).

Example 4 Radiolabelled Nanoformulations for Biological Assays

¹⁴C radiolabelled efavirenz samples were prepared in order to carry outcell accumulation and transwell (i.e. transcellular permeability)pharmacology studies. The samples were prepared based on the hits fromthe previous two screens (i.e. Example 1 and 2), i.e. 66 hits (65 hitsplus one extra) of 60% (P):30% (S); and 77 hits of 75% (P):15% (S). Thesamples were prepared in triplicate; one cold batch for DLS analysis andtwo hot batches pharmacology study. Both the cold and hot samples wereprepared following the same samples preparations mentioned previously.However, in order to prepare hot actives, 25 μC of 14C labelled EFV inethanol was measured out. The solvent was allowed to evaporate away andthe stock solution of cold EFV was added to the remaining solid. i.e.286 mg in 28.6 ml of chloroform ((2×66)+(2×77) in 286×100 μlchloroform). The cold batches were then studied via DLS to determineboth z-average and zeta potential.

Example 5 Preparing Higher Loading Nanoformulations (7 HydrophilicPolymers, 7 Surfactants)

Samples were prepared containing a higher loading of efavirenz, usingthe hydrophilic polymers and surfactants listed below in Table 5A.

TABLE 5A Hydrophilic polymers and surfactants employed HydrophilicPolymer Surfactant PEG 1k Na Deoxycholate F68 Vit E-peg-succinate F127AOT Kollicoat Solutol HS PVA Tween 20 PVP k30 Tween 80 HPMC Hyamine

The hydrophilic polymers and surfactants were selected based on the hitsfrom the previous screens, focussing particularly on approved excipientsappearing on the current FDA CDER list. The above 7 hydrophilic polymersand 7 hydrophilic surfactants were also selected based on their abilityto provide successful formulations of efavirenz.

Samples are prepared using a 70 mgml⁻¹ stock solution of efavirenz (A)in chloroform, a 22.5 mgml⁻¹ of polymer (P) and a 22.5 mgml⁻¹ stocksolution of surfactant (S). Stock solutions are added in the followingproportion; 100 μl (A); 90 μl (P) 45 μl (S) and 265 μl of water,therefore solid mass ratio is; 70% (A), 20% (P) and 10% (S) in an 1:4oil to water (O/W) mix. The mixtures are the emulsified using a CovarisS2x for 30 seconds with a duty cycle of 20, an intensity of 10 and 500cycles/burst in frequency sweeping mode. After which, the samples wereimmediately cryogenically frozen. A matrix of 49 samples (comprised of 7different polymers and 7 surfactants) was prepared. Once all 49 sampleshad been prepared, they were lyophilised (Virtis benchtop K) for 42hours to leave a dry porous product, the samples were then sealed inindividual vials until analysis.

High Loading (70% Active) Screen

Immediately prior to analysis samples were dispersed by addition of 3.5ml of water. All analysis was carried out in the same way as describedearlier in Example 1.

FIG. 1 shows a 3-D bar chart representing the clear hits in grey, nearmisses in white with dots, more distant misses in transparent, andnon-hits without bars. The z-average particle size for each of the hits,near misses, and more distant misses is given on the vertical axis.

Example 6 High Loading (70% Active) Formulation Variation

The formulation space around ‘hits’ (i.e. grey bars) from the highloading screen of Example 5 was further investigated. Here, thesurfactant to polymer ratio was varied at four levels shown in table 6Abelow.

TABLE 6A Table showing variation in polymer/surfactant ratio Polymersolution Surfactant solution Polymer (%) Surfactant (%) volume (μl)volume (μl) 25 5 112.5 22.5 20 10 90 45 15 15 67.5 67.5 10 20 45 90

Samples are prepared using a 70 mgml⁻¹ stock solution of efavirenz (A)in chloroform, a 22.5 mgml⁻¹ of polymer (P) and a 22.5 mgml⁻¹ stocksolution of surfactant (S). Stock solutions are added in the followingproportion; 100 μl (A), 265 μl of water, with the volumes of polymer andsurfactant solutions shown in the table above. All other preparation wasthe same as described in Example 5. The samples were then screened inthe same manner as Example 5, and again hits are given in grey in FIG.2.

FIG. 2 shows a screen of the 6 hits from Example 5 using variableproportions of polymer/surfactant

Table 6B below provides further details of screen, the single hit of thePVA/T-20 combination (which includes 20% surfactant), and a hit for eachof the other combinations at a 5% surfactant loading (since lowersurfactant quantities are desirable from a toxicity point of view).

TABLE 6B Details of 6 hits from the Example 6 screening experiments %Z-Av Z-Av Z-Av SD % Re- Surfactant Original Repeat AV from AV MadePVA/Vit E 5% 306.3 338.0 322.2 7.0 10% S PVA/T-20 20% 511.8 N — PVA/T-805% 370.3 378.5 374.4 1.5 10% S PVA/ 5% 430.5 509.0 469.7 11.8 10% SHyamine Kollicoat/ 5% 566.6 N — NDC PVA/NDC 5% 548.4 520.4 534.4 3.7 10%S [N = sample did not disperse, so size data not collected]

As can be seen from Table 6A, two of the repeat samples, namely PVA/T-20and Kollicoat/NDC, did not quite meet the above-described screeningcriteria, and therefore only 4 of the successful repeats were used forfurther experimentation.

FIG. 3 shows (a) the z-average particle size (nm) for the original andrepeat samples of each of the 4 successful repeat samples; and (b) theaverage zeta potential (mV) for each of the 4 successful repeat samples.

Table 6C shows analytical data for the 4 successful repeat samples,specifically the low % surfactant present (5% in all cases), z-averageparticle size of the original sample, z-average particle size of therepeat sample, average z-average particle size of both original andrepeat, % standard deviation (SD) from the average, % surfactant in afurther remade sample containing a high % of surfactant (10% in allcases), and the z-average particle size of the further high 10surfactant sample.

TABLE 6C analytical data for the 4 successful repeat samples Low % Z-AvZ-Av SD % High % Surfactant Original Repeat Z-Av AV from AV Surfactant ZAv PVA/Vit E 5% 306.3 338.0 322.2 7.0 10% S 292.9333 PVA/T-80 5% 370.3378.5 374.4 1.5 10% S 298.1667 PVA/ 5% 430.5 509.0 469.7 11.8 10% S426.9333 Hyamine PVA/NDC 5% 548.4 520.4 534.4 3.7 10% S 526.4333

FIG. 4 shows (a) the z-average particle size (nm) for low surfactant(5%) and high surfactant (10%) samples detailed in Table 6C; and (b) theaverage zeta potential (mV) for low surfactant (5%) and high surfactant(10%) samples detailed in Table 6C.

Example 7 High Loading Radiolabelled Nanoformulations for BiologicalAssays

From the above data in Example 6, 3 particular polymer/surfactantcombinations were selected for further study. The three samples wereselected on the basis that they showed good sample-to-samplereproducibility (i.e. a standard deviation below 10%). Furtherradiolabelled samples were prepared for the combinations of particularinterest, namely PVA/Vit-E, PVA/T-80, and PVA/NDC.

Radiolabelled efavirenz samples were prepared in accordance with Example4, however, the chloroform solution containing the efavirenz was alsodosed with the radiolabelled active to give a tracer concentration ofradioactivity for efavirenz samples. The samples were then used for allcellular accumulation and transcellular permeability studies (seeExamples 12 and 13).

Table 7A shows the z-average particle size (nm) and zeta potential (mV)analytical data in relation to original non-radiolabelled samplescontaining low (5%) and high (10%) surfactant concentrations, and alsoradiolabelled samples containing low (5%) and high (10%) surfactantconcentrations.

TABLE 7A analytical data for non-radiolabelled (original) andradiolabelled samples Original Samples Radiolabelled Samples 5%Surfactant 10% Surfactant 5% Surfactant 10% Surfactant EFV Z-Av ZetaZ-Av Zeta Z-Av Zeta Z-Av Zeta PVA/Vit-E 322.2 −22.1333 292.9 −20.4 363.2−19.5 346.4 −21.1 PVA/T-80 374.4 −16.5667 298.2 −23 343.3 −16.1 385−25.1 PVA/NDC 534.4 −17.7667 526.4 −15.0667 578.4 −20.2 765.9 −19.9

Nanodispersions of the cold (i.e. non-radiolabelled) samples were thenanalysed over time with respect to their z-average particle size,particle size polydispersity (i.e. diversity of particle sizes), andzeta potential.

FIG. 5A shows time course measurements made upon nanodispersions of thecold PVA/Vit-E samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/Vit-Esamples comprising both 5% (left hand side) and 10% (right hand size)surfactant content.

FIG. 5B shows time course measurements made upon nanodispersions of thecold PVA/T-80 samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/T-80samples comprising both 5% (left hand side) and 10% (right hand size)surfactant content.

FIG. 5C shows time course measurements made upon nanodispersions of thecold PVA/NDC samples (for both 5% and 10% surfactant samples) withrespect to their (a) z-average particle size (nm); (b) particle sizepolydispersity; and (c) average zeta potential (mV), each for PVA/NDCsamples comprising both 5% (left hand side) and 10% (right hand size)surfactant content.

FIGS. 5A-5C demonstrate the stability of nanodispersions of efavirenz ofthe present invention over prolonged time periods.

Example 8 Active Loading Studies

A study to measure the observed variation in particle size was carriedout by preparing samples were by addition of 100 μl efavirenz solutionsof increased concentrations, thereby increasing active loading. The oilto water ratio remained 1:4 throughout. The efavirenz concentrationsused were 10, 20, 30 40 and 50 mgml⁻¹, to which were added to 267 μl ofpolymer solution and 133 μA surfactant solution (each 22.5 mgml⁻¹).Therefore, for each set of exipients, 5 points are measured. Excipientswere chosen due to their good reproducibility qualities. Polymers; Peg1k, Kollicoat, PVA and Hydrolysed gelatine (H-Gel); Surfactants; NDC,T-20, Brij 58, Hyamine. Therefore 16 formulations in total (4×4combinations of polymer and surfactant), each with 5 activeconcentration points.

FIG. 6 below shows the increase in particle z-average with increasedefavirenz loading. The data shows three lines; crosses are total numberof hits including those which failed the DLS size quality criteria;solid triangles are the total hits of those where all data passed DLScriteria (7 in total where all 5 points hit); solid squares are thepredicted size based on volume of a sphere. The data for each line isquite consistent with the predicted values.

Example 9 Cytotoxicity of Efavirenz Nanodispersions in Hepatic,Intestinal, Monocyte, Macrophage and Lymphocyte Cell Lines

Routine Cell Culture/Cell Maintenance

HepG2 (hepatic) and Caco-2 (intestinal) cells were purchased fromAmerican Type Tissue Culture (ATCC; USA) and maintained in Dulbecco'smodified eagle's medium (DMEM; Sigma; Dorest, Uk) supplemented with 10%fetal bovine serum (FBS; Bio-Whittaker, Berkshire, Uk) for HepG2 and 15%FBS for Caco-2. CEM (lymphocyte) and THP-1 (monocyte) cells werepurchased from European Collection of Cell Culture (ECACC; Porton Down,Uk) and grown in RPMI-1640 (Sigma; Dorest, Uk) supplemented with 10%FBS. All cell lines were incubated at 37° C. and 5% CO₂ and for adherentcells (HepG2 and Caco-2) were routinely sub-cultured every 4 days when95% confluent. Suspension cells (CEM and THP-1) were routinelysub-cultured when a density of 1×10⁶ cells/ml was achieved. Cell countand viability was determined by Trypan Blue exclusion assay.

Monocyte Derived Macrophages (MDM)

THP-1 cells were activated into macrophage like cells (A-THP-1) by theaddition of Phorbol 12-myristate 13 acetate (PMA: Sigma; Dorest, Uk) tofinal concentration of 10 nM in THP-1 culture medium (RPMI-1640supplemented with 10% FBS). Cells were then incubated at 37° C. and 5%CO₂ for 7 days prior to use to allow differentiation from monocyte tomacrophage like cells.

HepG2, Caco-2, THP-1, CEM and A-THP-1 cells were separately seeded at adensity of 2.5×10⁴/100 μl in their appropriate media into each well of a96 well plate (Nunclon™, Denmark) and incubated for 24 hours at 37° C.and 5% CO₂. Media was then aspirated and replaced with media containing0.1, 1, 10, 100, 500 or 1000 μM, calculated from molarity of efavirenz(145 samples), of each efavirenz dispersion and incubated for a further24 hours. The same procedure was followed for each individual excipient(23 samples) with concentrations range of 1 μM to 1 M.

ATP Cell Viability Assay (Promega: CellTiter-Glo® Luminescent)

Prior to starting viability assays, all reagents were made fresh and inaccordance to manufacturer's instructions and allowed to equilibrate toroom temperature immediately before use. After 24 hours incubation, 96well plates were removed from the incubator and the plate and itscontents allowed too equilibrate to room temperature for approximately30 minutes. After 30 minutes, 80 μl of media was aspirated from eachindividual well and 20 μl CellTiter-Glo® Reagent (Promega, UK) was addedto the remaining media and cells. The contents were then mixed for 2minutes on an orbital shaker to induce cell lysis. The plate was thenfurther incubated at room temperature for 10 minutes to stabiliseluminescent signal. Luminescence was then measured using a Tecan Geniosplate reader (Tecan; Austria).

FIG. 7 provides data in relation to the cytotoxicity of the various 10%drug loaded efavirenz formulations of Examples 1 and 2 in terms of IC₅₀(μM) across various cell lines. Data for aqueous efavirenz (i.e.non-nanodisperse) are given as dotted horizontal lines.

A set of nanodispersions was selected from the various 70% drug loadedefavirenz formulations of Example 5 for further study, details of whichare outlined in Table 8 below. All the nanodispersion listed in Table 8have a drug loading of 70%, and a combined polymer/surfactant loading of30% (as in Example 5), but the surfactant and surfactant loading isvaried in each case. A control aqueous solution is also included forcomparative purposes.

TABLE 8 Details of particular nanodispersions selected for furtherstudy. Zeta % Particle potential Designation Polymer SurfactantSurfactant size (Z-av) (mV) Nanodispersion 1 PVA Vit E  5% 363 −20Nanodispersion 2 PVA T80  5% 343 −16 Nanodispersion 3 PVA NDC  5% 578−20 Nanodispersion 4 PVA Vit E 10% 346 −21 Nanodispersion 5 PVA T80 10%385 −25 Nanodispersion 6 PVA NDC 10% 766 −20 Aq. Solution n/a n/a n/an/a n/a (control)

Table 9 provides data in relation to the cytotoxicity of the various 70%drug loaded efavirenz formulations listed in Table 8 in terms of IC₅₀(μM).

TABLE 9 Cytotoxicity of 70% efavirenz loaded nanodispersions and aqueousefavirenz across various cell lines. Data are given as IC₅₀ in μM. Cellline Designation HepG2 Caco-2 THP-1 A-THP-1 CEM Nanodispersion 1 90 12171 148 169 Nanodispersion 2 96 111 93 92 177 Nanodispersion 3 61 47 4850 73 Nanodispersion 4 67 98 65 112 139 Nanodispersion 5 72 96 81 87 155Nanodispersion 6 43 39 33 43 80 Aqueous solution 33 39 29 29 40

In summary, the large majority of the 10% loaded nanodispersions wereless cytotoxic than equivalent concentrations of aqueous efavirenz. For70% loaded efavirenz nanodispersions, all were less cytotoxic thanaqueous efavirenz in the studied cell systems.

Example 10 Inhibition of Recombinant HIV-1 Reverse Transcriptase byNanodispersed and Aqueous Efavirenz

HIV-1 reverse transcriptase (RT) inhibition screening was conducted asper manufacturers' instructions (Roche Diagnostics GmbH; Germany).Briefly, for each of the individual nano dispersed EFV formulations, 20μl of 1 nM stock was added to each well of row B of a 96 well plate((Nunclon™, Denmark). Doubling dilutions were then preformed usingdeionised H₂O to give a range of concentrations from 1 nM to 0.0078 nMfor each sample. 20 μl of 0.2 ng/μl HIV-1-RT diluted in lysis buffer wasthen added to each well. 20 μl of reaction mixture (consisting of; 46 mMTris-HCl, 266 mM potassium chloride, 27.5 mM magnesium chloride, 9.2 mMDTT, 10 μM dUTP/dTTP, template/primer hybrid, 750 mA_(260nm)/ml) wasadded to each well containing EFV sample and the plate incubated for 60minutes at 37° C. The entire sample (60 μl) was then transferred intomicroplate modules and incubated for a further 60 minutes at 37° C. Thesamples were then aspirated and washed ×5 with 250 μl of wash buffer.After the final aspiration, 200 μl of reconstitutedanti-digoxigenin-peroxidase (Anti-DIG-POD) polyclonal antibody (200mU/ml final concentration) was added to all wells and the plate returnedto the incubator for a further 60 minutes at 37° C. The samples wereaspirated and washed ×5 with wash buffer. Finally, after the final washbuffer was aspirated, 200 μl of ABTS substrate was added to each welland the plate incubated at room temperature for 15 minutes. Absorbancewas then measured at 405 nm using FLUOstar Omega microplate reader (BMGLabtech, Uk).

The above procedure allowed for determination of the activity of the 10%drug loaded (FIG. 8) efavirenz nanodispersions (from Examples 1 and 2)and 70% drug loaded (Table 10) efavirenz nanodispersions (from Table 8)against the target protein, reverse transcriptase.

FIG. 8 shows activity of 10% loaded efavirenz nanodispersions againstrecombinant HIV reverse transcriptase. Data for aqueous efavirenz aregiven as the dotted horizontal line. Again, each circle represents aresult for a particular nanodispersion. Data are given as IC₅₀ in nM.

Table 10 gives IC₅₀ values for the 70% drug loaded (table 10) efavirenznanodispersions (from Table 8) along with the control aqueous solutionagainst the target protein, reverse transcriptase. Data are given asIC₅₀ in nM.

TABLE 10 Activity of 70% loaded efavirenz nanodispersions againstrecombinant HIV reverse transcriptase. Designation IC₅₀ (nM)Nanodispersion 1 154 Nanodispersion 2 223 Nanodispersion 3 331Nanodispersion 4 112 Nanodispersion 5 131 Nanodispersion 6 274 Aqueoussolution 123

In summary, most of the 10% loaded nanodispersions exhibited equivalentactivity to aqueous efavirenz for inhibition of recombinant HIV reversetranscriptase. 18 of the 145 10% loaded nanodispersions were more potentinhibitors. For 70% loaded efavirenz nanodispersions, all were able toinhibit recombinant reverse transcriptase within the nM range. 3 of the6 70% loaded nanodispersions exhibited equivalent or better activityagainst recombinant HIV reverse transcriptase. Data should beinterpreted in light of this being a cell-free assay.

Example 11 Inhibition of Cultured HIV-IIIB by Nanodispersed and AqueousEfavirenz in MT4 Cells

MT4/HIVIII_(B) Routine Cell Culture/Propagation

HIVIII_(B) and the human T-cell line, MT4, were supplied by the NationalInstitute for Biological Standards and Control (NIBSC), the Centre forAIDS Reagents (CFAR: Hertfordshire, Uk). MT4 cells were grown androutinely maintained in RPMI-1640 (Sigma; Dorest, Uk) supplemented with10% fetal bovine serum (FBS; Bio-Whittaker, Berkshire, Uk) at 37° C. and5% CO₂ and sub-cultured every 3 days. Cell counts and viability wasdetermined by Trypan Blue exclusion using a disposable plastichaemocytometer (Labtech International Ltd, UK). HIVIII_(B) waspropagated by passage through MT4 cells in RPMI-1640 and 10% FBS for 12days and a subsequent multiplicity of infection (MOI) was determined byMTT assay.

Treatment of HIVIII_(B) with Nanodispersed EFV

MT4 cells were dispensed into 50 ml falcon tubes and centrifuged at 2000rpm and 4° C. for 5 minutes. The supernatant was aspirated and theresulting pellet resuspended to a cell density of 1×10⁵/ml in RPMI 1640supplemented with 10% FBS and 1 ml of viral isolate (M.O.I 0.001). Afterfurther centrifugation for 5 minutes at 2000 rpm, the MT4/HIVIII_(B)pellet was incubated at 37° C. and 5% CO₂ for 2 hours. Followingincubation, the MT4/HIVIII_(B) suspension was centrifuged again at 2000rpm and 4° C. for 5 minutes, supernatant aspirated and pellet resuspenedRPMI 1640 supplemented with 10% FBS to a cell density of 1×10⁵/ml.

100 μl of MT4/HIVIII_(B) suspension was then added to each well of aflat bottomed 96 well plate (Nunclon™, Denmark) containing each of theindividual nano dispersed Efavirenz (EFV) formulations to give a rangeof final concentrations from 1 nM to 0.0078 nM. The plate was thenincubated for 5 days at 37° C. and 5% CO₂.

Tetrazolium-Based Colorimetric (MTT) Assay Using MT4 Cells for theDetection of Anti-HIV Compounds

Following incubation for 5 days of MT4/HIVIII_(B) with nano formulatedEFV at 37° C. and 5% CO₂. 20 μl of 5 mg Thiazolyl Blue Tetrazolium(Sigma, Uk) per ml of Hanks balanced salt solution (HBSS: Sigma, Uk) wasadded to each well and then further incubated for 2 hours at 37° C. and5% CO₂ 100 μl of lysis buffer containing 50% v/v dimethylformahyde(Sigma, Uk) and 20% v/v sodium dodecyl sulphate (Sigma, Uk) and incubateagain for 24 hours. Absorbance was read at 570 nm on a plate reader(Tecan Magellan, Austria).

The above procedures allowed for determination of the activity of the10% drug loaded (FIG. 9) and 70% drug loaded (table 11) efavirenznanodispersions against cultured HIV virus.

FIG. 9 shows activity of 10% loaded efavirenz nanodispersions againstrecombinant HIV reverse transcriptase. Data for aqueous efavirenz aregiven as the dotted horizontal line. Again, each circle represents aresult for a particular nanodispersion. Data are given as IC₅₀ in nM.

TABLE 11 Activity of 70% loaded efavirenz nanodispersions againstrecombinant HIV reverse transcriptase. Data are given as IC₅₀ in nM.Designation IC₅₀ (nM) Nanodispersion 1 42 Nanodispersion 2 25Nanodispersion 3 55 Nanodispersion 4 16 Nanodispersion 5 22Nanodispersion 6 76 Aqueous solution 71

In summary, most of the 10% loaded nanodispersions exhibited equivalentactivity higher than that of aqueous efavirenz for inhibition ofHIVIIIB. 127 of the 145 10% loaded nanodispersions were more potent thanaqueous efavirenz. For 70% loaded efavirenz nanodispersions, all wereable to inhibit recombinant reverse transcriptase within the nM rangeand all 7 exhibited equivalent or better activity against recombinantHIV reverse transcriptase. Data should be interpreted in light of thisbeing a cell-based assay (see example 13 for impact of nanoformulationon cellular accumulation).

Example 12 Cellular Accumulation of Nano Dispersed EFV Compared to theirParental, Aqueous Compounds

Cellular Accumulation of Nano Dispersed EFV Compared to their Parental,Aqueous Compounds in Adherent Cell Lines; Caco-2, HepG2 and ATHP-1.

Cellular accumulation of nano dispersed EFV was quantified in adherentcell lines, Caco-2 and HepG2 and ATHP-1's as follows. Caco-2/HepG2 werepropagated to a cell density of ˜5×10⁶ cells per well on a 6 well plate(37° C. and 5% CO₂). The cells were washed ×2 with pre-warmed HBSS (37°C.). The cells were incubated for 1 h in the presence of 1 ml of HBSScontaining 10 μM EFV containing 0.1 μCi of H³/C¹⁴/H³ labelled drugrespectively. After 1 hr, an extracellular sample was taken fromsupernatant (100 μl) and added into a 5 ml scintillation vial (MeridianBiotechnologies Ltd; Uk). The supernatant containing drug was aspiratedfrom the well and cells washed ×2 in ice cold HBSS. The ice cold HBSSwas aspirated and the addition of 500 μl of tap water to lyse the cells.The cellular debris plates (6 well) were stored overnight at −20° C. toaid lysis. The 500 μl of tap water was then used to pipette away celldebris from the well base and added to a 5 ml scintillation vial(Meridian Biotechnologies Ltd; Uk). To each 5 ml vial, 4 ml of UltimaGold scintillation fluid was added and scintillation level quantifiedusing a Perkin Elmeer 3100TS scintillation counter. Each nano-dispersionwas tested in duplicate and scintillation measurements taken intriplicate.

This culture method is altered for the ATHP-1 cell line as although theexperimental procedure for the accumulation is unaltered differentiationfrom monocyte to MDM (ATHP-1) cells must occur before the protocol isused. In brief, THP-1 cells, containing phorbol-12-myristate 13 acetate(PMA: Sigma; Dorest, Uk) to final concentration of 10 nM in medium(RPMI-1640 supplemented with 10% FBS) were seeded at a density ofapproximately 4×10⁶ cells per well on a 6 well plate. Cells were thenincubated at 37° C. and 5% CO₂ for 7 days prior to use to allowdifferentiation to macrophage like cells. Cellular accumulation of nanodispersed drug was quantified as previously described for Caco-2 andHepG2 cell lines.

Cellular Accumulation of Nano Dispersed EFV Compared to their Parental,Aqueous Compounds in Non-Adherent Cells; THP-1 and CEM

Cellular accumulation was ascertained for each nano-dispersion in THP-1and CEM cell lines as follows. In brief, cells were harvested at adensity of 5×10⁶ cells per well. Each well was washed ×2 with pre-warmedHBSS (37° C.). To each well, 600 μl of HBSS containing 10 μM EFV, eachwith 0.1 μCi of 3H/14C/3H labelled drug respectively. After incubationfor 1 hr the cells are pelleted by centrifugation (2K rpm, 5 min). After1 hr, an extracellular sample was taken from supernatant (100 μl) andadded into a 5 ml scintillation vial (Meridian Biotechnologies Ltd; Uk).The supernatant containing drug was aspirated from the well and cellswashed ×2 in ice cold HBSS. The ice cold HBSS was aspirated and theaddition of 100 μl of tap water to lyse the cells. The 100 μl of celllysate is then transferred to a 5 ml scintillation vial (MeridianBiotechnologies Ltd; Uk), to each 4 ml of Ultima Gold scintillationfluid was added and scintillation level quantified using a Perkin Elmer3100TS scintillation counter. Each nano-dispersion was tested induplicate and scintillation measurements taken in triplicate.

FIG. 10 shows cellular accumulation ratio (cell-associated drug dividedby extracellular drug) of all 10% loaded efavirenz nanodispersions inthe studied cell lines. Data for aqueous efavirenz are given as dottedhorizontal lines. Again, each circle represents a result for aparticular nanodispersion.

FIGS. 11A-E show cellular accumulation ratio (cell-associated drugdivided by extracellular drug) of 70% loaded efavirenz nanodispersionsin the studied cell lines, namely (A) HepG2, (B) Caco-2, (C) THP-1, (D)A-THP-1, (E) CEM, with respect to the six nanodispersed efavirenzformulations (detailed in Table 8) and the parental aqueous drug (i.e.non-nanodisperse) sample described in the present example.

In summary, 10% loaded nanodispersions which exhibit higher cellularpenetration into all cell types studied have been synthesised andcharacterised. In addition, 70% loaded nanodispersions with favourablepenetration into these cell types (particularly immune cells) have beenidentified.

Example 13 Transcellular Permeability of Efavirenz NanodispersionsAcross Caco-2 cell Monolayers

For transcellular permeability studies to identify 10% and 70% drugloaded nanodispersion candidates, modelling systemic circulation uptake,Caco-2 cells were propagated to a monolayer over a 21 day period,yielding transepithelial electrical resistance (TER) values of ˜1300Ω.For evaluation of 10% loaded nanoparticles, 10 μM of parental ornanoformulated drug (including 0.1 μCi radio-labelled drug) was added tothe apical chamber and samples taken at 1 and 2 hr from the basolateralchamber. Each nano dispersed drug was tested in duplicate. Forevaluation of 70% loaded nanodispersions, 10 μM of parental ornanoformulated drug (including 0.1 μCi radio-labelled drug) was added tothe apical chamber of 4 wells and the basolateral chamber of 4 wells toquantify transport in both apical to basolateral and basolateral toapical direction and sampled on an hourly basis over a 4 h time period.Apparent permeability coefficient was determined by the amount ofcompound transported using the following equation: Papp=(dQ/dt)(1/(AC₀). Where (dQ/dt) is the amount per time (nmol. sec⁻¹), A is thesurface area of the filter and C₀ is the starting concentration of thedonor chamber (10 μM).

FIG. 12 shows apical (gut compartment) to basolateral (bloodcompartment) transcellular permeability across Caco-2 cell monolayers of10% loaded efavirenz nanodispersions in the studied cell lines. Data foraqueous efavirenz are given as dotted horizontal lines. Again, eachcircle represents a result for a particular nanodispersion.

FIGS. 13A-F show transcellular permeability of 70% efavirenz loadednanodispersions across Caco-2 cell monolayers relative to aqueousefavirenz. Data are given for apical to basolateral and for basolateralto apical passage of drug. Each of panels A-F respectively representNanodispersions 1-6 detailed in Table 8 above.

Example 14 Pharmacokinetics of Efavirenz in Rodents Administered aNanodispersion Versus a Conventional Formulation

Adult male Wistar rats, (250-350 g) were terminally anesthetized with14% urethane in 0.9% saline (1 ml/100 g body weight i.p) and cannulatedvia the trachea and carotid artery. A pre dose blood sample 0.1 ml wascollected into a heparinised Eppendorf vial and centrifuged at 10,000rpm for 5 minutes. The plasma layer was then removed and stored at −20C. Rats were then orally dosed with efavirenz or an efavirenznanodispersion using a 7 cm curved gavarge needle 0.1 ml blood sampleswere collected via the carotid cannula at the following time points 20′,30′, 45′, 60′, 90′, 120′, 180′, 240′, 300′. At the end of the experimentanimals were killed using excess of anesthetic. 200 μl of a 30% Hydrogenperoxide (Sigma-Aldrich, Uk) solution was added to each sample and leftfor 60 minutes. Finally, 30 μl of glacial acetic acid was added and theentire sample incubated for a further 15 minutes at 50° C. 4 ml ofUltima Gold (PerkinElmer, Uk) scintillation fluid was added, samplemixed vigorously, and the radioactivity in Disintegrations per Minute(dpm) was counted using a 3100 TR liquid scintillation counter (IsoTech,Uk). Plasma concentrations of efavirenz were then calculated from thedpm.

FIG. 14 shows pharmacokinetics of efavirenz and 70%-loaded efavirenznanodispersion in rats. A demonstrably higher plasma efavirenzconcentration was seen for the nanodispersion than for a conventionalformulation of efavirenz.

CONCLUSION

The efavirenz formulations of the present invention are hereby shown toform stable nanodispersions with a number of favourable pharmacologicalproperties. Nanodispersions have been synthesised that have lowercytotoxicity, that exhibit more potent inhibition of HIV reversetranscriptase, that exhibit more potent inhibition of HIV replication,that accumulate to a higher degree in HIV target cells and that traverseintestinal epithelial cells more rapidly and more completely thanaqueous solutions of efavirenz. Furthermore, nanodispersions ofefavirenz with favourable pharmacokinetic properties relative toconventional formulation have been generated.

ABBREVIATIONS

Na alginate=sodium alginate

Na Myristate=sodium myristate

Na Deoxycholate=sodium deoxycholate

Na Caprylate=sodium caprylate

Vit E-peg-succinate=D-α-Tocopherol polyethylene glycol 1000 succinate

Sisterna 11=Sisterna SP50

Sisterna 16=Sisterna PS750

SDS=Sodium dodecyl sulphate

AOT=Dioctyl sodium sulfosuccinate

Solutol HS=Solutol HS 15

Hyamine=_Benzethonium chloride

CTAB=Cetrimonium bromide

PEG=polyethylene glycol

Kollicoat=Kollicoat Protect (trade name)

PVA=Polyvinyl acetate

PVP=Polyvinylpyrrolidone

HPMC=Hydroxypropyl methylcellulose

NaCMC=sodium carboxymethylcellulose

What is claimed is:
 1. A solid efavirenz composition, comprising solidnanoparticles of efavirenz dispersed within a solid mixture of at leastone hydrophilic polymer and at least one surfactant; wherein thehydrophilic polymer is selected from polyvinyl alcohol (PVA), apolyvinyl alcohol-polyethylene glycol graft copolymer, a block copolymerof polyoxyethylene and polyoxypropylene, polyethylene glycol,hydroxypropyl methyl cellulose (HPMC), and polyvinylpyrrolidone, or acombination thereof; and the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), apolyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammoniumchloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA), and ablock copolymer of polyoxyethylene and polyoxypropylene, or acombination thereof.
 2. A solid efavirenz composition according to claim1, wherein the solid nanoparticles of efavirenz have an average particlesize of less than or equal to 1 micron (μm).
 3. A solid efavirenzcomposition according to claim 2, wherein the solid nanoparticles ofefavirenz have an average particle size between 100 and 600 nm.
 4. Asolid efavirenz composition according to claim 1, wherein the solidnanoparticles of efavirenz have a polydispersity of less than or equalto 0.8.
 5. A solid efavirenz composition according to claim 1, whereinthe hydrophilic polymer is selected from PVA, Poloxamer 407, PEG 1K,HPMC, PVP K30, and Poloxamer 188, or a combination thereof.
 6. A solidefavirenz composition according to claim 5, wherein the hydrophilicpolymer is selected from PVA or a polyvinyl alcohol-polyethylene glycolgraft copolymer.
 7. A solid efavirenz composition according to claim 6,wherein the hydrophilic polymer is PVA.
 8. A solid efavirenz compositionaccording to claim 1, wherein the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), apolyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammoniumchloride, sodium deoxycholate, dioctyl sodium sulfosuccinate, andpolyethyleneglycol-12-hydroxystearate, or a combination thereof.
 9. Asolid efavirenz composition according to claim 8, wherein the surfactantis selected from vitamin-E-polyethylene glycol-succinate(Vit-E-PEG-succinate), Polysorbate 20, Polysorbate 80,N-alkyldimethylbenzylammonium chloride, sodium deoxycholate, and dioctylsodium sulfosuccinate.
 10. A solid efavirenz composition according toclaim 9, wherein the surfactant is selected from vitamin-E-polyethyleneglycol-succinate (Vit-E-PEG-succinate), Polysorbate 20, Polysorbate 80,N-alkyldimethylbenzylammonium chloride, and sodium deoxycholate.
 11. Asolid efavirenz composition according to claim 1, wherein: (i) thehydrophilic polymer is PVA and the surfactant is selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate),Polysorbate 20, Polysorbate 80, N-alkyldimethylbenzylammonium chloride,sodium deoxycholate, and dioctyl sodium sulfosuccinate; or (ii) thehydrophilic polymer is polyvinyl alcohol-polyethylene glycol graftcopolymer and the surfactant is sodium deoxycholate.
 12. Apharmaceutical composition in a solid dosage form comprising a solidcomposition according to claim 1, and optionally one or more additionalpharmaceutically acceptable excipients.
 13. A solid compositionaccording to claim 1 for use in the treatment or delaying the appearanceof clinical symptoms of a retrovirus infection wherein the solidcomposition is administered in combination with one or more otherantiretroviral agents.
 14. A pharmaceutical composition according toclaim 12 for use in the treatment or prevention of a retrovirusinfection wherein the pharmaceutical composition is administered incombination with one or more other antiretroviral agents.
 15. A processfor preparing a solid efavirenz composition according to claim 1, theprocess comprising: milling a solid form of efavirenz in the presence ofat least one hydrophilic polymer selected from polyvinyl alcohol (PVA),a polyvinyl alcohol-polyethylene glycol graft copolymer, a blockcopolymer of polyoxyethylene and polyoxypropylene, polyethylene glycol,hydroxypropyl methyl cellulose (HPMC), and polyvinylpyrrolidone, or acombination thereof, and at least one surfactant selected fromvitamin-E-polyethylene glycol-succinate (Vit-E-PEG-succinate), apolyoxyethylene sorbitan fatty acid ester, N-alkyldimethylbenzylammoniumchloride, sodium deoxycholate, dioctyl sodium sulfosuccinate,polyethyleneglycol-12-hydroxystearate, polyvinyl alcohol (PVA), and ablock copolymer of polyoxyethylene and polyoxypropylene, or acombination thereof.